Mitotic kinesin inhibitors

ABSTRACT

The present invention relates to dihydropyrrole compounds that are useful for treating cellular proliferative diseases, for treating disorders associated with KSP kinesin activity, and for inhibiting KSP kinesin. The invention is also related to compositions which comprise these compounds, and methods of using them to treat cancer in mammals.

BACKGROUND OF THE INVENTION

This invention relates to 2,2-disubstituted 2,5-dihydropyrrolederivatives that are inhibitors of mitotic kinesins, in particular themitotic kinesin KSP, and are useful in the treatment of cellularproliferative diseases, for example cancer, hyperplasias, restenosis,cardiac hypertrophy, immune disorders and inflammation.

Among the therapeutic agents used to treat cancer are the taxanes andvinca alkaloids. Taxanes and vinca alkaloids act on microtubules, whichare present in a variety of cellular structures. Microtubules are theprimary structural element of the mitotic spindle. The mitotic spindleis responsible for distribution of replicate copies of the genome toeach of the two daughter cells that result from cell division. It ispresumed that disruption of the mitotic spindle by these drugs resultsin inhibition of cancer cell division, and induction of cancer celldeath. However, microtubules form other types of cellular structures,including tracks for intracellular transport in nerve processes. Becausethese agents do not specifically target mitotic spindles, they have sideeffects that limit their usefulness.

Improvements in the specificity of agents used to treat cancer is ofconsiderable interest because of the therapeutic benefits which would berealized if the side effects associated with the administration of theseagents could be reduced. Traditionally, dramatic improvements in thetreatment of cancer are associated with identification of therapeuticagents acting through novel mechanisms. Examples of this include notonly the taxanes, but also the camptothecin class of topoisomerase Iinhibitors. From both of these perspectives, mitotic kinesins areattractive targets for new anti-cancer agents.

Mitotic linesins are enzymes essential for assembly and function of themitotic spindle, but are not generally part of other microtubulestructures, such as in nerve processes. Mitotic linesins play essentialroles during all phases of mitosis. These enzymes are “molecular motors”that transform energy released by hydrolysis of Al? into mechanicalforce which drives the directional movement of cellular cargoes alongmicrotubules. The catalytic domain sufficient for this task is a compactstructure of approximately 340 amino acids. During mitosis, kinesinsorganize microtubules into the bipolar structure that is the mitoticspindle. Kinesins mediate movement of chromosomes along spindlemicrotubules, as well as structural changes in the mitotic spindleassociated with specific phases of mitosis. Experimental perturbation ofmitotic kinesin function causes malformation or dysfunction of themitotic spindle, frequently resulting in cell cycle arrest and celldeath.

Among the mitotic kinesins which have been identified is KSP. KSPbelongs to an evolutionarily conserved kinesin subfamily of plusend-directed microtubule motors that assemble into bipolar homotetramersconsisting of antiparallel homodimers. During mitosis KSP associateswith microtubules of the mitotic spindle. Microinjection of antibodiesdirected against KSP into human cells prevents spindle pole separationduring prometaphase, giving rise to monopolar spindles and causingmitotic arrest and induction of programmed cell death. KSP and relatedkinesins in other, non-human, organisms, bundle antiparallelmicrotubules and slide them relative to one another, thus forcing thetwo spindle poles apart. KSP may also mediate in anaphase B spindleelongation and focussing of microtubules at the spindle pole.

Human KSP (also termed HsEg5) has been described [Blangy, et al., Cell,83:1159-69 (1995); Whitehead, et al., Arthritis Rheum., 39:1635-42(1996); Galgio et al., J. Cell Biol., 135:339-414 (1996); Blangy, etal., J Biol. Chem., 272:19418-24 (1997); Blangy, et al., Cell MotilCytoskeleton, 40:174-82 (1998); Whitehead and Rattner, J. Cell Sci.,111:2551-61 (1998); Kaiser, et al., JBC 274:18925-31 (1999); GenBankaccession numbers: X85137, NM004523 and U37426], and a fragment of theKSP gene (TRIP5) has been described [Lee, et al., Mol Endocrinol.,9:243-54 (1995); GenBank accession number L40372]. Xenopus KSP homologs(Eg5), as well as Drosophila K-LP61 F/KRP 130 have been reported.

Certain quinazolinones have recently been described as being inhibitorsof KSP (PC Publ. WO 01/30768, May 3, 2001).

Mitotic kinesins are attractive targets for the discovery anddevelopment of novel mitotic chemotherapeutics. Accordingly, it is anobject of the present invention to provide compounds, methods andcompositions useful in the inhibition of KSP, a mitotic kinesin.

SUMMARY OF THE INVENTION

The present invention relates to dihydropyrrole derivatives, that areuseful for treating cellular proliferative diseases, for treatingdisorders associated with KSP kinesin activity, and for inhibiting KSPkinesin. The compounds of the invention may be illustrated by theFormula I:

DETAILED DESCRIPTION OF THE INVENTION

The compounds of this invention are useful in the inhibition of mitotickinesins and are Illustrated by a compound of Formula I:

-   or a pharmaceutically acceptable salt or stereoisomer thereof,-   wherein:-   a is 0 or 1;-   b is 0 or 1;-   m is 0, 1, or 2;-   n is 0, 1, 2 or 3;-   r is 0 or 1;-   s is 0 or 1;-   t is 0, 1 or 2;-   R¹ and R² are independently selected from: H, (C₁-C₆)alkyl, aryl,    heterocyclyl and (C₃-C₆)cycloalkyl, optionally substituted with one,    two or three substituents selected from R⁷;-   R³ is selected from

1) hydrogen;

2) C₁-C_(10 alkyl;)

3) C₁-C₁₀ alkyl-O—R^(d),

4) C₂-C₁₀ alkenyl-O—R^(d),

5) C₂-C₁₀ alkenyl-O—R^(d),

6) (C₁-C₆-alkylene)_(n)C₃-C₈ cycloalkyl-O—R^(d),

7) C₁-C₁₀ alkyl-(C═O)_(b)—NR^(c)R^(c)′,

8) C₂-C₁₀ alkenyl-(C═O)_(b)NR^(c)R^(c)′,

9) C₂-C₁₀ alkynyl-(C═O)_(b)NR^(c)R^(c)′,

10) (C₁-C₆-alkylene)_(n)C₃-C₈ cycloalkyl-(C═O)_(b)NR^(c)R^(c)′,

11) C₁-C₁₀ alkyl-S(O)_(m)—R^(d),

12) C₂-C₁₀ alkenyl-S(O)_(m)—R^(d),

13) C₂-C₁₀ alkynyl-S(O)_(m)—R^(d),

14) (C₁-C₆-alkylene)_(n)C₃-C₈ cycloalkyl-S(O)_(m)—R^(d),

-   said alkyl, alkenyl, alkynyl and cycloalkyl are optionally    substituted with one or more substituents selected from R⁶;-   R⁴ is independently selected from:

1) (C═O)_(a)O_(b)C₁-C_(10 alkyl,)

2) (C═O)_(a)O_(b)aryl,

3) CO₂H,

4) halo,

5) CN,

6) OH,

7) O_(b)C₁-C₆ perfluoroalkyl,

8) O_(a)(C═O)_(b)NR⁸R⁹,

9) S(O)_(m)R^(a),

10) S(O)₂NR⁸R⁹,

-   said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl    optionally substituted with one, two or three substituents selected    from R⁷;-   R⁵ is selected from:

1) hydrogen;

2) (C═O)_(a)O_(b)C₁-C₁₀ alkyl,

3) (C═O)_(a)O_(b)aryl,

4) CO2H,

5) halo,

6) CN,

7) OH,

8) O_(b)C₁-C₆ perfluoroalkyl,

9) O_(a)(C═O)_(b)NR⁸R⁹,

10) S(O)_(m)R^(a),

11) S(O)₂NR⁸R⁹,

-   said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl    optionally substituted with one, two or three substituents selected    from R⁷;-   R⁶ is independently selected from:

1) (C═O)_(a)O_(b)C₁-C_(10 alkyl,)

2) (C═O)_(a)O_(b)aryl,

3) C₂-C₁₀ alkenyl,

4) C₂-C₁₀ alkynyl,

5) (C═O)_(a)O_(b) heterocyclyl,

7) halo,

8) CN,

9) OH,

10) O_(b)C₁-C₆ perfluoroalkyl

11) O_(a)(C═O)_(b)NR⁸R⁹,

12) S(O)_(m)R^(a),

13) S(O)₂NR⁸R⁹,

14) oxo,

15) CHO,

16) (N═O)R⁸R⁹, or

17) (C═O)_(a)O_(b)C₃-C₈ cycloalkyl,

-   said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl    optionally substituted with one, two or three substituents selected    from R⁷;-   R⁷ is selected from:

1) (C═O)_(r)O_(s)(C₁-C₁₀)alkyl ,

2) O_(r)(C₁-C₃)perfluoroalkyl,

3) oxo,

4) OH,

5) halo,

6) CN,

7) (C₂-C₁₀)alkenyl,

8) (C₂-C₁₀)alkyl,

9) (C═O)_(r)O_(s)(C₃-C₆)cycloalkyl,

10) (C═O)_(r)O_(s)(C₀-C₆)alkylene-aryl,

11) (C═O)_(r)O_(s)(C₀-C₆)alkylene-heterocyclyl,

12) (C═O)_(r)O_(s)(C₀-C₆)alkylene-N(R^(b))₂,

13) C(O)R^(a),

14) (C₀-C₆)alkylene-CO₂R^(a),

15) C(O)H,

16) (C₀-C₆)alkylene-CO₂H, and

17) C(O)N(R^(b))₂,

18) S(O)_(m)R^(a), and

19) S(O)₂N(R^(b))₂;

-   said alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylene and    heterocyclyl is optionally substituted with up to three substituents    selected from R^(b), OH, (C₁-C₆)alkoxy, halogen, CO₂H, CN,    O(C═O)C₁-C₆ alkyl, oxo, NO₂ and N(R^(b))₂;-   R⁸ and R⁹ are independently selected from:

1) H,

2) (C═O)O_(b)C₁-C₁₀ alkyl,

3) (C═O)O_(b)C₃-C₈ cycloalkyl,

4) (C═O)O_(b)aryl,

5) (C═O)O_(b)heterocyclyl,

6) C₁-C₁₀ alkyl,

7) aryl,

8) C₂-C₁₀ alkenyl,

9) C₂-C₁₀ alkynyl,

10) heterocyclyl,

11) C₃-C₈ cycloalkyl,

12) SO₂R^(a), and

13) (C═O)NR^(b) ₂,

-   said alkyl, cycloalkyl, aryl, heterocylyl, alkenyl, and alkynyl is    optionally substituted with one, two or three substituents selected    from R⁷, or-   R⁸ and R⁹ can be taken together with the nitrogen to which they are    attached to form a monocyclic or bicyclic heterocycle with 3-7    members in each ring and optionally containing, in addition to the    nitrogen, one or two additional heteroatoms selected from N, O and    S, said monocyclic or bicyclic heterocycle optionally substituted    with one, two or three substituents selected from R⁷;-   R¹⁰ is selected from: F and —CH₂F;-   R¹¹ and R¹² are independently selected from: H and —CH₂F;-   R^(ox) is absent or is oxo;-   R^(a) is independently selected from: (C₁-C₆)alkyl,    (C₃-C₆)cycloalkyl, aryl, or heterocyclyl, optionally substituted    with one, two or three substituents selected from R⁷;-   R^(b) is independently selected from: H, (C₁-C₆)alkyl, aryl,    heterocyclyl, (C₃-C₆)cycloalkyl, (C═O)OC₁-C₆alkyl, (C═O)C₁-C₆ alkyl,    (C═O)aryl, (C═O)heterocyclyl, (C═O)NR^(e)R^(e) or S(O)₂R^(a),    optionally substituted with one, two or three substituents selected    from R⁷;-   R^(c) and R^(c)′ are independently selected from H, (C₁-C₆)alkyl,    aryl, NH₂, OH, OR^(a), —C₁-C₆)alkyl-OH,    —(C₁-C₆)alkyl-O—(C₁-C₆)alkyl, (C═O)OC₁-C₆ alkyl, (C═O)C₁-C₆ alkyl,    (C═O)aryl, (C═O)heterocyclyl, (C═O)NR^(e)R^(e)′, S(O)₂R^(a) and    —C₁-C₆)alkyl-N(R^(b))₂, wherein the alkyl is optionally substituted    with one, two or three substituents selected from R⁷; or-   R^(c) and R^(c)′ can be taken together with the nitrogen to which    they are attached to form a monocyclic or bicyclic heterocycle with    3-7 members in each ring and optionally containing, in addition to    the nitrogen, one or two additional heteroatoms selected from N, O    and S, said monocyclic or bicyclic heterocycle optionally    substituted with one, two or three substituents selected from R⁷;-   R^(d) is selected from: H, (C₁-C₆)alkyl, —(C₂-C₆)alkyl-OH,    —C₁-C₆)alkyl-O—(C₁-C₆)alkyl and —(C₁-C₆)alkyl-N(R^(b))₂, wherein the    allyl is optionally substituted with one, two or three substituents    selected from R⁷;-   R^(e) and R^(e)′ are independently selected from; H, (C₁-C₆)alkyl,    aryl, heterocyclyl and (C₃-C₆)cycloalkyl, optionally substituted    with one, two or three substituents selected from R⁷; or-   R^(e) and R^(e)′ can be taken together with the nitrogen to which    they are attached to form a monocyclic or bicyclic heterocycle with    3-7 members in each ring and optionally containing, in addition to    the nitrogen, one or two additional heteroatoms selected from N, O    and S, said monocyclic or bicyclic heterocycle optionally    substituted with one, two or three substituents selected from R⁷.

The compounds of this invention are useful in the inhibition of mitotickinesins and are illustrated by a compound of Formula II:

-   or a pharmaceutically acceptable salt or stereoisomer thereof,-   wherein:-   a is 0 or 1;-   b is 0 or 1;-   m is 0, 1, or 2;-   n is 0, 1, 2 or 3;-   r is 0 or 1;-   s is 0 or 1;-   t is 0 or 1;-   R¹ and R² are independently selected from: H, (C₁-C₆)alkyl, aryl,    heterocyclyl and (C₃-C₆)cycloalkyl, optionally substituted with one,    two or three substituents selected from R⁷;-   R³ is selected from:

1) hydrogen;

2) C₁-C₁₀ alkyl;

3) C₁-C₁₀ alkyl-O—R^(d),

4) C₂-C₁₀ alkenyl-O—R^(d),

5) C₂-C₁₀ alkynyl-O—R^(d),

6) (C₁-C₆-alkylene)_(n)C₃-C₈ cycloalkyl-O—R^(d),

7) C₁-C₁₀ alkyl-C═O)_(b)—NR^(c)R^(c)′,

8) C₂-C₁₀ alkenyl-(C═O)_(b)NR^(c)R^(c)′,

9) C₂-C₁₀ alkynyl-(C═O)_(b)NR^(c)R^(c)′,

10) (C₁-C₆-alkylene)_(n)C₃-C₈ cycloalkyl(C═O)_(b)NR^(c)R^(c)′,

11) C₁-C₁₀ alkyl-S(O)_(m)—R^(d),

12) C₂-C₁₀ alkenyl-S(O)_(m)—R^(d),

13) C₂-C₁₀ alkynyl-S(O)_(m)—R^(d),

14) (C₁-C₆-alkylene)_(n)C₃-C₈ cycloalkyl-S(O)_(m)—R^(d),

-   said alkyl, alkenyl, alkynyl and cycloalkyl are optionally    substituted with one or more substituents selected from R⁶;-   R⁴ is independently selected from.

1) (C═O)_(a)O_(b)C₁-C₁₀ alkyl,

2) (C-O)_(a)O_(b)aryl,

3) CO₂H,

4) halo,

5) CN,

6) OH,

7) O_(b)C₁-C₆ perfluoroalkyl,

8) O_(a)(C═O)_(b)NR⁸R⁹,

9) S(O)_(m)R^(a),

10) S(O)₂NR⁸R⁹,

-   said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl    optionally substituted with one, two or three substituents selected    from R⁷;-   R⁵ is selected from:

1) hydrogen;

2) (C═O)_(a)O_(b)C₁-C₁₀ alkyl,

3) (C═O)_(a)O_(b)aryl,

4) CO₂H,

5) halo,

6) CN,

7) OH,

8) O_(b)C₁-C₆ perfluoroalkyl,

9) O_(a)(C═O)_(b)NR⁸R⁹,

10) S(O)_(m)R^(a),

11) S(O)₂NR⁸R⁹,

-   said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl    optionally substituted with one, two or three substituents selected    from R⁷;-   R⁶ is independently selected from:

1) (C═O)_(a)O_(b)C₁-C₁₀ alkyl,

2) (C═O)_(a)O_(b)aryl,

3) C₂-C₁₀ alkenyl,

4) C₂-C₁₀ alkynyl,

5) (C═O)_(a)O_(b) heterocyclyl,

6) CO₂H,

7) halo,

8) CN,

9) OH,

10) O_(b)C₁-C₆ perfluoroalkyl,

11) O_(a)(C═O)_(b)NR⁸R⁹,

12) S(O)_(m)R^(a),

13) S(O)₂NR⁸R⁹,

14) oxo,

15) CHO,

16) (N═O)R⁸R⁹, or

17) (C═O)_(a)O_(b)C₃-C₈ cycloalkyl,

-   said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl    optionally substituted with one, two or three substituents selected    from R⁷;-   R⁷ is selected from:

1) (C═O)_(r)O_(s)(C₁-C₁₀)alkyl,

2) O_(r)(C₁-C₃)perfluoroalkyl,

3) oxo,

4) OH,

5) halo,

6) CN,

7) (C₂-C₁₀)alkenyl,

8) (C₂-C₁₀)alkynyl,

9) (C═O)_(r)O_(s)(C₃-C₆)cycloalkyl,

10) (C═O)_(r)O_(s)(C₀-C₆)alkylene-aryl,

11) (C═O)_(r)O_(s)(C₀-C₆)alkylene-heterocyclyl,

12) (C═O)_(r)O_(s)(C₀-C₆)alkylene-N(R^(b))₂,

13) C(O)R^(a),

14) (C₀-C₆)alkylene-CO₂R^(a),

15) C(O)H,

16) (C₀-C₆)alkylene-CO₂H, and

17) C(O)N(R^(b))₂,

18) S(O)_(m)R^(a), and

19) S(O)₂N(R^(b))₂;

-   said alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylene and    heterocyclyl is optionally substituted with up to three substituents    selected from R^(b), OH, (C₁-C₆)alkoxy, halogen, CO₂H, CN,    O(C═O)C₁-C₆ alkyl, oxo, NO₂ and N(R^(b))₂;-   R⁸ and R⁹ are independently selected from:

1) H,

2) (C═O)O_(b)C₁-C₁₀ alkyl,

3) (C═O)O_(b)C₃-C₈ cycloalkyl,

4) (C═O)O_(b)aryl,

5) (C═O)O_(b)heterocyclyl,

6) C₁-C₁₀ alkyl,

7) aryl,

8) C₂-C₁₀ alkenyl,

9) C₂-C₁₀ alkynyl,

10) heterocyclyl,

11) C₃-C₈ cycloalkyl,

12) SO₂R^(a), and

13) (C═O)NR^(b) ₂,

-   said alkyl, cycloalkyl, aryl, heterocylyl, alkenyl, and alkynyl is    optionally substituted with one, two or three substituents selected    from R⁷, or-   R⁸ and R⁹ can be taken together with the nitrogen to which they are    attached to form a monocyclic or bicyclic heterocycle with 3-7    members in each ring and optionally containing, in addition to the    nitrogen, one or two additional heteroatoms selected from N, O and    S, said monocyclic or bicyclic heterocycle optionally substituted    with one, two or three substituents selected from R⁷;-   R¹⁰ is selected from: F and —CH₂F;-   R¹² is selected from: H and —CH₂F, provided that when t is 1, R¹² is    H;-   R^(ox) is absent or is oxo;-   R^(a) is independently selected from; (C₁-C₆)alkyl,    (C₃-C₆)cycloalkyl, aryl, or heterocyclyl, optionally substituted    with one, two or three substituents selected from R⁷;-   R^(b) is independently selected from: H, (C₁-C₆)alkyl, aryl,    heterocyclyl, (C₃-C₆)cycloalkyl, (C═O)OC₁-C₆ alkyl, (C═O)C₁-C₆    alkyl, (C═O)aryl, (C═O)heterocyclyl, (C═O)NR^(e)R^(e)′ or    S(O)₂R^(a), optionally substituted with one, two or three    substituents selected from R⁷;-   R^(c) and R^(c)′ are independently selected from: H, (C₁-C₆)alkyl,    aryl, NH₂, OH, OR^(a), —(C₁-C₆)alkyl-OH,    —(C₁-C₆)alkyl-O—(C₁-C₆)alkyl, (C═O)OC₁-C₆ alkyl, (C═O)C₁-C₆ alkyl,    (C═O)aryl, (C═O)heterocyclyl, (C═O)NR^(e)R^(e)′, S(O)₂R^(a) and    —(C₁-C₆)alkyl-N(R^(b))₂, wherein the alkyl is optionally substituted    with one, two or three substituents selected from R⁷; or-   R^(c) and R^(c)′ can be taken together with the nitrogen to which    they are attached to form a monocyclic or bicyclic heterocycle with    3-7 members in each ring and optionally containing, in addition to    the nitrogen, one or two additional heteroatoms selected from N, O    and S, said monocyclic or bicyclic heterocycle optionally    substituted with one, two or three substituents selected from R⁷;-   R^(d) is selected from: H, (C₁-C₆₎alkyl, —(C₂-C₆)alkyl-OH,    —(C₁-C₆)alkyl-O—C₁-C₆)alkyl and —(C₁-C₆)alkyl-N(R^(b))₂, wherein the    alkyl is optionally substituted with one, two or three substituents    selected from R⁷;;-   R^(e) and R^(e)′ are independently selected from H, (C₁-C₆)alkyl,    aryl, heterocyclyl and (C₃-C₆)cycloalkyl, optionally substituted    with one, two or three substituents selected from R⁷; or-   R^(e) and R^(e)′ can be taken together with the nitrogen to which    they are attached to form a monocyclic or bicyclic heterocycle with    3-7 members in each ring and optionally containing, in addition to    the nitrogen, one or two additional heteroatoms selected from N, O    and S, said monocyclic or bicyclic heterocycle optionally    substituted with one, two or three substituents selected from R⁷.

In an embodiment of the invention the compounds are illustrated by acompound of Formula III:

-   or a pharmaceutically acceptable salt or stereoisomer thereof,-   wherein:

a is 0 or 1;

b is 0 or 1;

m is 0, 1, or 2;

n is 0, 1 or 2;

r is 0 or 1;

s is 0 or 1;

-   R¹ and R² are independently selected from: H, (C₁-C₆)alkyl, aryl and    (C₃-C₆)cycloalkyl, optionally substituted with one, two or three    substituents selected from R⁷;-   R⁴ is independently selected from:

1) halo,

2) OH,

3) O_(b)C₁-C₆ perfluoroalkyl,

R⁵ is selected from:

1) hydrogen,

2) halo,

3) OH,

4) O_(b)C₁-C₆ perfluoroalkyl,

-   R⁷ is selected from:

1) (C═O)_(r)O_(s)(C₁-C₁₀)alkyl,

2) O_(r)(C₁-C₃)perfluoroalkyl,

3) oxo,

4) OH,

5) halo,

6) CN,

7) (C₂-C₁₀)alkenyl,

8) (C₂-C₁₀)alkynyl,

9) (C═O)_(r)O_(s)(C₃-C₆)cycloalkyl,

10) (C═O)_(r)O_(s)(C₀-C₆)alkylene-aryl,

11) (C═O)_(r)O_(s)(C₀-C₆)alkylene-heterocyclyl,

12) (C-O)_(r)O_(s)(C₀-C₆)alkylene-N(R^(b))₂,

13) C(O)R^(a),

14) (C₀-C₆)alkylene-CO₂R^(a),

15) C(O)H,

16) (C₀-C₆)alkylene-CO₂H, and

17) C(O)N(R^(b))₂,

18) S(O)N(R^(a), and

19) S(O)₂N(R^(b))₂;

-   said allyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylene and    heterocyclyl is optionally substituted with up to three substituents    selected from R^(b), OH, (C₁-C₆)alkoxy, halogen, CO₂H, CN,    O(C═O)C₁-C₆ alkyl, oxo, NO₂ and N(R^(b))₂;-   R⁸ and R⁹ are independently selected from:

1) H,

2) (C═O)O_(b)C₁-C₁₀ alkyl,

3) (C═O)O_(b)C₃-C₈ cycloalkyl,

4) (C═O)O_(b)aryl,

5) (C═O)O_(b)heterocyclyl,

6) C₁-C₁₀ alkyl,

7) aryl,

8) C₂-C₁₀ alkenyl,

9) C₂-C₁₀ alkyl,

10) heterocyclyl,

11) C₃-C₈ cycloalkyl,

12) SO₂R^(a), and

13) (C═O)NR^(b) ₂,

-   said alkyl, cycloalkyl, aryl, heterocylyl, alkenyl, and alkynyl is    optionally substituted with one, two or three substituents selected    from R⁷, or-   R⁸ and R⁹ can be taken together with the nitrogen to which they are    attached to form a monocyclic or bicyclic heterocycle with 3-7    members in each ring and optionally containing, in addition to the    nitrogen, one or two additional heteroatoms selected from N, O and    S, said monocyclic or bicyclic heterocycle optionally substituted    with one, two or three substituents selected from R⁷;-   R^(a) is independently selected from: (C₁-C₆)alkyl,    (C₃-C₆)cycloalkyl, aryl, or heterocyclyl, optionally substituted    with one, two or three substituents selected from R⁷;-   R^(b) is independently selected from: H, (C₁-C₆)alkyl, aryl,    heterocyclyl, (C₃-C₆)cycloalkyl, (C═O)OC₁-C₆ alkyl, (C═O)C₁-C₆    alkyl, (C═O)aryl, (C═O)heterocyclyl, (C═O)NR^(e)R^(e)′ or    S(O)₂R^(a), optionally substituted with one, two or three    substituents selected from R⁷;-   R^(c) and R^(c)′ are independently selected from: H, (C₁-C₆)alkyl,    aryl, NH₂, OH, OR^(a), —C₁-C₆)alkyl-OH, —(C₁-C₆)alkyl-O—C₁-C₆)alkyl,    (C═O)OC₁-C₆ alkyl, (C═O)C₁-C₆ alkyl, (C═O)aryl, (C═O)heterocyclyl,    (C═O)NR^(e)R^(e)′, S(O)₂R^(a) and —C₁-C₆)alkyl-N(R)₂, wherein the    alkyl is optionally substituted with one, two or three substituents    selected from R⁷; or-   R^(c) and R^(c)′ can be taken together with the nitrogen to which    they are attached to form a monocyclic or bicyclic heterocycle with    3-7 members in each ring and optionally containing, in addition to    the nitrogen, one or two additional heteroatoms selected from N, O    and S, said monocyclic or bicyclic heterocycle optionally    substituted with one, two or three substituents selected from R⁷;-   R^(e) and R^(e)′are independently selected from: H. (C₁-C₆)alkyl,    aryl, heterocyclyl and (C₃-C₆)cycloalkyl, optionally substituted    with one, two or three substituents selected from R⁷; or-   R^(e) and R^(e)′ can be taken together with the nitrogen to which    they are attached to form a monocyclic or bicyclic heterocycle with    3-7 members in each ring and optionally containing, in addition to    the nitrogen, one or two additional heteroatoms selected from N, O    and S, said monocyclic or bicyclic heterocycle optionally    substituted with one, two or three substituents selected from R⁷.

Another embodiment of the present invention is illustrated by a compoundof Formula IV:

-   or a pharmaceutically acceptable salt or stereoisomer thereof,-   wherein:-   a is 0 or 1;-   b is 0 or 1;-   m is 0, 1, or 2;-   r is 0 or 1;-   s is 0 or 1;-   R¹ and R² are independently selected from H and (C₁-C₆)alkyl,    optionally substituted with one, two or three substituents selected    from R⁷;-   R⁴ is independently selected from:

1) halo,

2) OH,

3) O_(b)C₁-C₆ perfluoroalkyl,

-   R⁷ is selected from:

1) (C═O)_(r)OS(C₁-C₁₀)alkyl,

2) O_(r)(C₁-C₃)perfluoroalkyl,

3) oxo,

4) OH,

5) halo,

6) CN,

7) (C₂-C₁₀)alkenyl,

8) (C₂-C₁₀)alkynyl,

9) (C═O)_(r)O_(s)(C₃-C₆)cycloalkyl,

10) (C═O)_(r)O_(s)(C₀-C₆)alkylene-aryl,

11) (C═O)_(r)O_(s)(C₀-C₆)alkylene-heterocyclyl,

12) (C═O)_(r)O_(s)(C₀-C₆)alkylene-N(R^(b))₂,

13) C(O)R^(a),

14) (C₀-C₆)alkylene-CO₂R^(a),

15) C(O)H,

16) (C₀-C₆)alkylene-CO₂H, and

17) C(O)N(R^(b))₂,

18) S(O)_(m)R^(a), and

19) S(O)₂N(R^(b))₂;

-   said alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylene and    heterocyclyl is optionally substituted with up to three substituents    selected from R^(b), OH, (C₁-C₆)alkoxy, halogen, CO₂H, CN,    O(C═O)C₁-C₆ alkyl, oxo, NO₂ and N(R^(b))₂;-   R⁸ and R⁹ are independently selected from:

1) H,

2) (C═O)O_(b)C₁-C₁₀ alkyl,

3) (C-O)O_(b)C₃-C₈ cycloalkyl,

4) (C═O)O_(b)aryl,

5) (C═O)O_(b)heterocyclyl,

6) C₁-C₁₀ alkyl,

7) aryl,

8) C₂-C₁₀ alkenyl,

9) C₂-C₁₀ alkynyl,

10) heterocyclyl,

11) C₃-C₈ cycloalkyl,

12) SO₂R^(a), and

13) (C═O)NR^(b) ₂,

-   said alkyl, cycloalkyl, aryl, heterocylyl, alkenyl, and alkynyl is    optionally substituted with one, two or three substituents selected    from R⁷, or-   R⁸ and R⁹ can be taken together with the nitrogen to which they are    attached to form a monocyclic or bicyclic heterocycle with 3-7    members in each ring and optionally containing, in addition to the    nitrogen, one or two additional heteroatoms selected from N, O and    S, said monocyclic or bicyclic heterocycle optionally substituted    with one, two or three substituents selected from R⁷;-   R^(a) is independently selected from: (C₁-C₆)alkyl,    (C₃-C₆)cycloalkyl, aryl, or heterocyclyl, optionally substituted    with one, two or three substituents selected from R⁷;-   R^(b) is independently selected from: H, (C₁-C₆)alkyl, aryl,    heterocyclyl, (C₃-C₆)cycloalkyl, (C═O)OC₁-C₆ allyl, (C═O)C₁-C₆    alkyl, (C═O)aryl, (C═O)heterocyclyl, (C═O)NR^(e)R^(e)′ or    S(O)₂R^(a), optionally substituted with one, two or three    substituents selected from R⁷;-   R^(c) and R^(c)′ are independently selected from H, (C₁-C₆)alkyl,    aryl, NH₂, OH, OR^(a), —(C₁-C₆)alkyl-OH, —(C₁-C₆)alkyl-OC₁-C₆)alkyl,    (C═O)OC₁-C₆ alkyl, (C═O)C₁-C₆ alkyl, (C═O)aryl, (C═O)heterocyclyl,    (C═O)NR^(e)R^(e)′, S(O)₂R^(a) and —(C₁-C₆)alkyl-N(R^(b))₂, wherein    the alkyl is optionally substituted with one, two or three    substituents selected from R⁷; or-   R^(c) and R^(c)′ can be taken together with the nitrogen to which    they are attached to form a monocyclic or bicyclic heterocycle with    3-7 members in each ring and optionally containing, in addition to    the nitrogen, one or two additional heteroatoms selected from N, O    and S, said monocyclic or bicyclic heterocycle optionally    substituted with one, two or three substituents selected from R⁷;-   R^(e) and R^(e)′ are independently selected from: H, (C₁-C₆)alkyl,    aryl, heterocyclyl and (C₃-C₆)cycloalkyl, optionally substituted    with one, two or three substituents selected from R⁷; or-   R^(e) and R^(e)′ can be taken together with the nitrogen to which    they are attached to form a monocyclic or bicyclic heterocycle with    3-7 members in each ring and optionally containing, in addition to    the nitrogen, one or two additional heteroatoms selected from N, O    and S, said monocyclic or bicyclic heterocycle optionally    substituted with one, two or three substituents selected from R⁷.

Specific examples of the compounds of the instant invention include:

-   (2S)-4-(2,5-Difluorophenyl)-N-[(3R,4R)-4-fluoropyrrolidin-3-yl]-2-(hydroxymethyl)N-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide-   (2S)-4-(2,5-Difluorophenyl)-N-[(3 S,4S)-4-fluoropyrrolidin-3    -yl]-2-(hydroxymethyl)N-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide-   (2S)-4-(2,5-Difluorophenyl)-N-[(3R,4R)-4-fluoro-1-methylpyrrolidin-3-yl]-2-(hydroxymethyl)N-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide-   (2S)-4-2,5-Difluorophenyl)-N-[(3S,4S)-4-fluoro-1-methylpyrrolidin-3-yl]-2-hydroxymethyl)N-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide-   (2S)-4-(2,5-Difluorophenyl)-N-[(3S,5S)-5-fluoromethyl)-pyrrolidin-3-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide-   (2S)-4-(2,5-Difluorophenyl)-N-[(3S,5S)-5-(fluoromethyl)-1-methylpyrrolidin-3-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide-   (2S)-4-(2,5-Difluorophenyl)-N-[(3S,5R)-5-fluoromethyl)-pyrrolidin-3-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide-   (2S)-4-(2,5-Difluorophenyl)-N-[(3S,5R)-5-(fluoromethyl)-1-methylpyrrolidin-3-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide-   or a pharmaceutically acceptable salt or stereoisomer thereof.

The compounds of the present invention may have asymmetric centers,chiral axes, and chiral planes (as described in: E. L. Eliel and S. H.Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York,1994, pages 1119-1190), and occur as racemates, racemic mixtures, and asindividual diastereomers, with all possible isomers and mixturesthereof, including optical isomers, all such stereoisomers beingincluded in the present invention. In addition, the compounds disclosedherein may exist as tautomers and both tautomeric forms are intended tobe encompassed by the scope of the invention, even though only onetautomeric structure is depicted.

When any variable (e.g. R⁴, R⁷, R¹⁰, etc.) occurs more than one time inany constituent, its definition on each occurrence is independent atevery other occurrence. Also, combinations of substituents and variablesare permissible only if such combinations result in stable compounds.Lines drawn into the ring systems from substituents represent that theindicated bond may be attached to any of the substitutable ring atoms.If the ring system is polycyclic, it is intended that the bond beattached to any of the suitable carbon atoms on the proximal ring only.

It is understood that substituents and substitution patterns on thecompounds of the instant invention can be selected by one of ordinaryskill in the art to provide compounds that are chemically stable andthat can be readily synthesized by techniques known in the art, as wellas those methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.The phrase “optionally substituted with one or more substituents” shouldbe taken to be equivalent to the phrase “optionally substituted with atleast one substituent” and in such cases the preferred embodiment willhave from zero to three substituents.

As used herein, “alkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms. For example, C₁-C₁₀, as in “C₁-C₁₀alkyl” is defined to include groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 carbons in a linear or branched arrangement. For example, “C₁-C₁₀alkyl” specifically includes methyl, ethyl, n-propyl, i-propyl, n-butyl,t-butyl, i-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and so on.The term “cycloalkyl” means a monocyclic saturated aliphatic hydrocarbongroup having the specified number of carbon atoms. For example,“cycloalkyl” includes cyclopropyl, methyl-cyclopropyl,2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, and so on. Inan embodiment of the invention the term “cycloalkyl” includes the groupsdescribed immediately above and further includes monocyclic unsaturatedaliphatic hydrocarbon groups. For example, “cycloalkyl” as defined inthis embodiment includes cyclopropyl, methyl-cyclopropyl,2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, cyclopentenyl,cyclobutenyl and so on.

The term “alkylene” means a hydrocarbon diradical group having thespecified number of carbon atoms. For example, “alkylene” includes—CH₂—, —CH₂CH₂— and the like.

When used in the phrases “C₁-C₆ akyl” and “C₁-C₆ heteroaralkyl” the term“C₁-C₆” refers to the alkyl portion of the moiety and does not describethe number of atoms in the aryl and heteroaryl portion of the moiety.

“Alkoxy” represents either a cyclic or non-cyclic alkyl group ofindicated number of carbon atoms attached through an oxygen bridge.“Alkoxy” therefore encompasses the definitions of alkyl and cycloalkylabove.

If no number of carbon atoms is specified, the term “alkenyl” refers toa non-aromatic hydrocarbon radical, straight, branched or cyclic,containing from 2 to 10 carbon atoms and at least one carbon to carbondouble bond. Preferably one carbon to carbon double bond is present, andup to four non-aromatic carbon-carbon double bonds may be present. Thus,“C₂-C₆ alkenyl” means an alkenyl radical having from 2 to 6 carbonatoms. Alkenyl groups include ethenyl, propenyl, butenyl,2-methylbutenyl and cyclohexenyl. The straight, branched or cyclicportion of the alkenyl group may contain double bonds and may besubstituted if a substituted alkenyl group is indicated.

The term “alkynyl” refers to a hydrocarbon radical straight, branched orcyclic, containing from 2 to 10 carbon atoms and at least one carbon tocarbon triple bond. Up to three carbon-carbon triple bonds may bepresent. Thus, “C₂-C₆ alkynyl” means an alkynyl radical having from 2 to6 carbon atoms. Alkynyl groups include ethynyl, propynyl, butynyl,3-methylbutynyl and so on. The straight, branched or cyclic portion ofthe alkynyl group may contain triple bonds and may be substituted if asubstituted alkynyl group is indicated.

In certain instances, substituents may be defined with a range ofcarbons that includes zero, such as (C₀-C₆)alkylene-aryl. If aryl istaken to be phenyl, this definition would include phenyl itself as wellas —CH₂Ph, —CH₂CH₂Ph, CH(CH₃)CH₂CH(CH₃)Ph, and so on.

As used herein, “aryl” is intended to mean any stable monocyclic orbicyclic carbon ring of up to 7 atoms in each ring, wherein at least onering is aromatic. Examples of such aryl elements include phenyl,naphthyl, tetrahydronaphthyl, indanyl and biphenyl. In cases where thearyl substituent is bicyclic and one ring is non-aromatic, it isunderstood that attachment is via the aromatic ring.

The term heteroaryl, as used herein, represents a stable monocyclic orbicyclic ring of up to 7 atoms in each ring, wherein at least one ringis aromatic and contains from 1 to 4 heteroatoms selected from the groupconsisting of O, N and S. Heteroaryl groups within the scope of thisdefinition include but are not limited to: acridinyl, carbazolyl,cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl,thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl,oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl,pyrimidinyl, pyrrolyl, tetrahydroquinoline. As with the definition ofheterocycle below, “heteroaryl” is also understood to include theN-oxide derivative of any nitrogen-containing heteroaryl. In cases wherethe heteroaryl substituent is bicyclic and one ring is non-aromatic orcontains no heteroatoms, it is understood that attachment is via thearomatic ring or via the heteroatom containing ring, respectively.

The term “heterocycle” or “heterocyclyl” as used herein is intended tomean a 5- to 10-membered aromatic or nonaromatic heterocycle containingfrom 1 to 4 heteroatoms selected from the group consisting of O, N andS, and includes bicyclic groups. “Heterocyclyl” therefore includes theabove mentioned heteroaryls, as well as dihydro and tetrathydro analogsthereof. Further examples of “heterocyclyl” include, but are not limitedto the following: benzoimidazolyl, benzofuranyl, benzofurazanyl,benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl,carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl,indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl,oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl,pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl,pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl,tetrazolyl, tetrarolopyridyl, thiadiazolyl, thiazolyl, thienyl,triazolyl, azetidinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl,piperidinyl, pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl,dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl,dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl,dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, andN-oxides thereof. Attachment of a heterocyclyl substituent can occur viaa carbon atom or via a heteroatom.

Preferably, heterocycle is selected from 2-azepinone, benzimidazolyl,2-diazapinone, imidazolyl, 2-imidazolidinone, indolyl, isoquinolinyl,morpholinyl, piperidyl, piperazinyl, pyridyl, pyrrolidinyl,2-piperidinone, 2-pyrimidinone, 2-pyrollidinone, quinolinyl,tetrahydrofuryl, tetrahydroisoquinolinyl, and thienyl.

As appreciated by those of skill in the art, “halo” or “halogen” as usedherein is intended to include chloro, fluoro, bromo and iodo.

The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl andheterocyclyl substituents may be substituted or unsubstituted, unlessspecifically defined otherwise. For example, a (C₁-C₆)alkyl may besubstituted with one, two or three substituents selected from OH, oxo,halogen, alkoxy, dialkylamino, or heterocyclyl, such as morpholinyl,piperidinyl, and so on. In this case, if one substituent is oxo and theother is OH, the following are included in the definition:—C═O)CH₂CH(OH)CH₃, —(C═O)OH, —CH₂(OH)CH₂CH(O), and so on.

In certain instances, R^(c) and R^(c)′ and R^(f) and R^(f)′ are definedsuch that they can be taken together with the nitrogen to which they areattached to form a monocyclic or bicyclic heterocycle with 5-7 membersin each ring and optionally containing, in addition to the nitrogen, oneor two additional heteroatoms selected from N, O and S, said heterocycleoptionally substituted with one or more substituents selected from R⁷.Examples of the heterocycles that can thus be formed include, but arenot limited to the following, keeping in mind that the heterocycle isoptionally substituted with one or more (and in an embodiment, one, twoor three) substituents chosen from R⁷:

In certain instances, R^(d) and R^(d)′ are defined such that they can betaken together with the phosphorous to which they are attached to form amonocyclic heterocycle with 5-7 members in the ring and optionallycontaining, in addition to the nitrogen, one or two additionalheteroatoms selected from NR^(e), O and S, said heterocycle optionallysubstituted with one or more substituents selected from R⁷. Examples ofthe heterocycles that can thus be formed include, but are not limited tothe following, keeping in mind that the heterocycle is optionallysubstituted with one or more (and in an embodiment, one or two)substituents chosen from R⁷:

In an embodiment, R¹ is selected from H and C₁-C₆ alkyl.

In an embodiment, R² is selected from H and C1-C₆ alkyl.

In an embodiment, R¹¹ and R¹² are H.

In an embodiment, R³ is selected from —C₁-C₁₀ alkyl-O—R^(g) and —C₁-C₁₀alkyl-NR^(f)R^(f)′, optionally substituted with one to two substituentsselected from R¹⁰.

In an embodiment, R⁴ is independently selected from halogen and OH. In afurther embodiment, n is 2 and R⁴ is independently selected fromhalogen.

In an embodiment, R⁵ is independently selected from H, halogen and OH.

In an embodiment, t is 1 and R¹⁰ is fluoro.

In another embodiment, t is 0 and R¹¹ is fluoromethyl.

In another embodiment, t is 0 and R¹² is fluoromethyl.

In an embodiment, R^(ox) is absent.

Included in the instant invention is the free form of compounds ofFormula I, as well as the pharmaceutically acceptable salts andstereoisomers thereof. Some of the specific compounds exemplified hereinare the protonated salts of amine compounds. The term “free form” refersto the amine compounds in non-salt form. The encompassedpharmaceutically acceptable salts not only include the salts exemplifiedfor the specific compounds described herein, but also all the typicalpharmaceutically acceptable salts of the free form of compounds ofFormula I. The free form of the specific salt compounds described may beisolated using techniques known in the art. For example, the free formmay be regenerated by treating the salt with a suitable dilute aqueousbase solution such as dilute aqueous NaOH, potassium carbonate, ammoniaand sodium bicarbonate. The free forms may differ from their respectivesalt forms somewhat in certain physical properties, such as solubilityin polar solvents, but the acid and base salts are otherwisepharmaceutically equivalent to their respective free forms for purposesof the invention.

The pharmaceutically acceptable salts of the instant compounds can besynthesized from the compounds of this invention which contain a basicor acidic moiety by conventional chemical methods. Generally, the saltsof the basic compounds are prepared either by ion exchangechromatography or by reacting the free base with stoichiometric amountsor with an excess of the desired salt-forming inorganic or organic acidin a suitable solvent or various combinations of solvents. Similarly,the salts of the acidic compounds are formed by reactions with theappropriate inorganic or organic base.

Thus, pharmaceutically acceptable salts of the compounds of thisinvention include the conventional non-toxic salts of the compounds ofthis invention as formed by reacting a basic instant compound with aninorganic or organic acid. For example, conventional non-toxic saltsinclude those derived from inorganic acids such as hydrochloric,hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like, aswell as salts prepared from organic acids such as acetic, propionic,succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic,pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic,salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic,methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroaceticand the like.

When the compound of the present invention is acidic, suitable“pharmaceutically acceptable salts” refers to salts prepared formpharmaceutically acceptable non-toxic bases including inorganic basesand organic bases. Salts derived from inorganic bases include aluminum,ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganicsalts, manganous, potassium, sodium, zinc and the like. Particularlypreferred are the ammonium, calcium, magnesium, potassium and sodiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as arginine, betainecaffeine, choline, N,N¹-dibenzylethylenediamine, diethylamin,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylaminetripropylamine, tromethamine and the like.

The preparation of the pharmaceutically acceptable salts described aboveand other typical pharmaceutically acceptable salts is more fullydescribed by Berg et al., “Pharmaceutical Salts,”0 J. Pharm. Sci.,1977:66:1-19.

It will also be noted that the compounds of the present invention arepotentially internal 20 salts or zwitterions, since under physiologicalconditions a deprotonated acidic moiety in the compound, such as acarboxyl group, may be anionic, and this electronic charge might then bebalanced off internally against the cationic charge of a protonated oralkylated basic moiety, such as a quaternary nitrogen atom.

The following abbreviations, used in the Schemes and Examples, aredefined below: CDI 1,1′-carbonyldiimidazole CSP HPLC Chiral stationaryphase high performance liquid chromatography DAST (diethylamino)sulfurtrifluoride DCE 1,2-dichloroethane DCM Dichloromethane DMFDimethylformamide DMSO Dimethyl sulfoxide EtOAc Ethyl acetate LAHLithium aluminum hydride LiHMDS Lithium hexamethyldisilazide MsClMethanesulfonylchloride NaHMDS Sodium hexamethyldisilazide NOE NuclearOverhauser Effect PTC Phase transfer catalyst TBSCltert-butyldimethylsilyl chloride TEA Triethylamine TFA Trifluoroaceticacid THF Tetrahydrofuran

The compounds of this invention may be prepared by employing reactionsas shown in the following schemes, in addition to other standardmanipulations that are known in the literature or exemplified in theexperimental procedures. The illustrative schemes below, therefore, arenot limited by the compounds listed or by any particular substituentsemployed for illustrative purposes. Substituent numbering as shown inthe schemes does not necessarily correlate to that used in the claimsand often, for clarity, a single substituent is shown attached to thecompound where multiple substituents are allowed under the definitionsof Formula I hereinabove.

Schemes

As shown in Scheme A, key 2,2-disubstituted dihydropyrrole intermediateA-8 may be obtained from readily available suitably substitutedα-phenylglycines. Following the procedure described by Van Betsbruggeet. al. (Tetrahedron, 1997, 53, 9233-9240) the α-allyl-α-phenylglycineis prepared. Reduction of the ester and cyclization withcarbonyldiimidazole provides intermediate A-4. Ruthenium oxidation ofthe allylic olefin, followed by ester formation and alkylation of thenitrogen provides intermediate A-5. Cyclization and decarboxylationresults in intermediate A6. The ring carbonyl can then be utilized toincorporate a suitably substituted phenyl moiety. Subsequentsaponification and oxygen protection leads to the protected intermediateA-8. The ring nitrogen may then be reacted with triphosgene to providethe activated intermediate A-9.

Scheme B illustrates the preparation of the fluorinated aminopyrrolidineBA from the epoxide B-1. Intermediate B-3 is described in Li, Q. et al,Bioorg. Med. Chenm. Lett. 8, 1998, 1953-1958. The aminopyrrolidine B4may then be reacted with intermediate A-9, as illustrated in Scheme C,to provide the bisprotected compound C-1. The acyclic nitrogen may bealkylated as shown. The intermediate C-2 may then be deprotected andring-nitrogen alkylated to provide the instant compound C-3.

Preparation of the fluorinated aminopyrrolidine D-2 having the cisorientation of the amine and the fluorine starting from the commonintermediate B-2 is shown in Scheme D. Preparation of theaminopyrrolidine intermediates having a fluoromethyl moiety in the5-position (E-5) or the 2-position (F-5) are illustrated in Schemes Eand F. These amines may be reacted with intermediate A-9 as illustratedin Scheme C above.

Scheme G illustrates the coupling of the2-fluoromethyl-3-aminopyrrolidine to intermediate A-9 followed bydeprotection and alkylation of the hydroxymethyl moiety on thedihydropyrrole ring.

Scheme H illustrates further elaboration of that hydroymethyl moiety toprepare the instant compounds H-2 and H-3.

The hydroxymethyl moiety may also be oxidized to the correspondingaldehyde I-1 which can then undergo reductive amination to provide theinstant compound I-2, as illustrated in Scheme I.

Use of alternative activating group on the amino carbonyl moiety isillustrated in Scheme J.

Scheme K illustrates further homologation of the ring hydroxymethylmoiety. Initially reaction with trimethyl pospohono acetate provides amixture of the unsaturated alcohol sidechain compound K-1 and thehomologous aldehyde K-2. These compounds may be chromatographicallyseparated and utilized to prepare the corresponding amines K-3 and K4.

The aldehyde I-1 may also be reacted with a Grignard reagent to providethe instant compound L-1, as illustrated in Scheme L.

As illustrated in Scheme M, the aldehyde moiety at the 2-position on thedihydropyrrole may also be treated with trimethylphosphonoacetate toprovide, after reduction of the conjugated double bond, the estercompound M-2. Lithium aluminum hydride reduction of M-2, followed byDess-Martin oxidation provide the aldehyde M-4, which can then bereductively aminated as previously described.

Scheme N illustrates fluorination of the C-2 sidechain and subsequentconversion of the hydroxyl moiety to an amine via displacement of thecorresponding triflate with sodium azide.

Scheme O illustrates incorporation of a difluoromethyl moiety into theC-2 sidechain.

Utilities

The compounds of the invention find use in a variety of applications. Aswill be appreciated by those skilled in the art, mitosis may be alteredin a variety of ways; that is, one can affect mitosis either byincreasing or decreasing the activity of a component in the mitoticpathway. Stated differently, mitosis may be affected (e.g., disrupted)by disturbing equilibrium, either by inhibiting or activating certaincomponents. Similar approaches may be used to alter meiosis.

In a preferred embodiment, the compounds of the invention are used tomodulate mitotic spindle formation, thus causing prolonged cell cyclearrest in mitosis. By “modulate” herein is meant altering mitoticspindle formation, including increasing and decreasing spindleformation. By “mitotic spindle formation” herein is meant organizationof microtubules into bipolar structures by mitotic kinesins. By “mitoticspindle dysfunction” herein is meant mitotic arrest and monopolarspindle formation.

The compounds of the invention are useful to bind to and/or modulate theactivity of a mitotic kinesin. In a preferred embodiment, the mitotickinesin is a member of the bimC subfamily of mitotic kinesins (asdescribed in U.S. Pat. No. 6,284,480, column 5). In a further preferredembodiment, the mitotic kinesin is human KSP, although the activity ofmitotic kinesins from other organisms may also be modulated by thecompounds of the present invention. In this context, modulate meanseither increasing or decreasing spindle pole separation, causingmalformation, i.e., splaying, of mitotic spindle poles, or otherwisecausing morphological perturbation of the mitotic spindle. Also includedwithin the definition of KSP for these purposes are variants and/orfragments of KSP. In addition, other mitotic kinesins may be inhibitedby the compounds of the present invention.

The compounds of the invention are used to treat cellular proliferationdiseases. Disease states which can be treated by the methods andcompositions provided herein include, but are not limited to, cancer(further discussed below), autoimmune disease, arthritis, graftrejection, inflammatory bowel disease, proliferation induced aftermedical procedures, including, but not limited to, surgery, angioplasty,and the like. It is appreciated that in some cases the cells may not bein a hyper- or hypoproliferation state (abnormal state) and stillrequire treatment. For example, during wound healing, the cells may beproliferating “normally”, but proliferation enhancement may be desired.Similarly, as discussed above, in the agriculture arena, cells may be ina “normal” state, but proliferation modulation may be desired to enhancea crop by directly enhancing growth of a crop, or by inhibiting thegrowth of a plant or organism which adversely affects the crop. Thus, inone embodiment, the invention herein includes application to cells orindividuals afflicted or impending affliction with any one of thesedisorders or states.

The compounds, compositions and methods provided herein are particularlydeemed useful for the treatment of cancer including solid tumors such asskin, breast, brain, cervical carcinomas, testicular carcinomas, etc.More particularly, cancers that may be treated by the compounds,compositions and methods of the invention include, but are not limitedto: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma,liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung:bronchogenic carcinoma (squamous cell, undifferentiated small cell,undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar)carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatoushamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cellcarcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach(carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma,insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), smallbowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformans), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma [pinealoma], glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma),fallopian tubes (carcinoma); Hematoloric: blood (myeloid leukemia [acuteand chronic], acute lymphoblastic leukemia, chronic lymphocyticleukemia, myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignantlymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma.Thus, the term “cancerous cell” as provided herein, includes a cellafflicted by any one of the above-identified conditions.

The compounds of the instant invention may also be useful as antifungalagents, by modulating the activity of the fungal members of the bimCkinesin subgroup, as is described in U.S. Pat. No. 6,284,480.

The compounds of the invention are also useful in preparing a medicamentthat is useful in treating the cellular proliferation diseases describedabove, in particular cancer.

The compounds of this invention may be administered to mammals,preferably humans, either alone or, preferably, in combination withpharmaceutically acceptable carriers, excipients or diluents, in apharmaceutical composition, according to standard pharmaceuticalpractice. The compounds can be administered orally or parenterally,including the intravenous, intramuscular, intraperitoneal, subcutaneous,rectal and topical routes of administration.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specific amounts, aswell as any product which results, directly or indirectly, fromcombination of the specific ingredients in the specified amounts.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavoring agents, coloring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients whichare suitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example, microcrystalline cellulose, sodiumcrosscarmellose, corn starch, or alginic acid; binding agents, forexample starch, gelatin, polyvinyl-pyrrolidone or acacia, andlubricating agents, for example, magnesium stearate, stearic acid ortalc. The tablets may be uncoated or they may be coated by knowntechniques to mask the unpleasant taste of the drug or delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a watersoluble taste masking material such as hydroxypropyl-methylcellulose orhydroxypropylcellulose, or a time delay material such as ethylcellulose, cellulose acetate butyrate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with watersoluble carrier such as polyethyleneglycol or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active material in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethyl-elrulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl phydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose, saccharin or aspartame.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as butylated hydroxyanisol or alpha-tocopherol.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present. These compositions may be preserved by theaddition of an anti-oxidant such as ascorbic acid.

The pharmaceutical compositions of the invention may also be in the formof an oil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally occurring phosphatides, for example soy bean lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening, flavoring agents, preservatives and antioxidants.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative, flavoring and coloring agentsand antioxidant.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous solutions. Among the acceptable vehicles and solventsthat may be employed are water, Ringer's solution and isotonic sodiumchloride solution.

The sterile injectable preparation may also be a sterile injectableoil-in-water microemulsion where the active ingredient is dissolved inthe oily phase. For example, the active ingredient may be firstdissolved in a mixture of soybean oil and lecithin. The oil solutionthen introduced into a water and glycerol mixture and processed to forma microemulation.

The injectable solutions or microemulsions may be introduced into apatient's blood stream by local bolus injection. Alternatively, it maybe advantageous to administer the solution or microemulsion in such away as to maintain a constant circulating concentration of the instantcompound. In order to maintain such a constant concentration, acontinuous intravenous delivery device may be utilized. An example ofsuch a device is the Deltec CADD-PLUS™ model 5400 intravenous pump.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension for intramuscular andsubcutaneous administration. This suspension may be formulated accordingto the known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example as a solution in 1,3-butane diol. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid find use in the preparation of injectables.

Compounds of Formula I may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials include cocoa butter, glycerinated gelatin,hydrogenated vegetable oils, mixtures of polyethylene glycols of variousmolecular weights and fatty acid esters of polyethylene glycol.

For topical use, creams, ointments, jellies, solutions or suspensions,etc., containing the compound of Formula I are employed. (For purposesof this application, topical application shall include mouth washes andgargles.)

The compounds for the present invention can be administered inintranasal form via topical use of suitable intranasal vehicles anddelivery devices, or via transdermal routes, using those forms oftransdermal slin patches well known to those of ordinary skill in theart. To be administered in the form of a transdermal delivery system,the dosage administration will, of course, be continuous rather thanintermittent throughout the dosage regimen. Compounds of the presentinvention may also be delivered as a suppository employing bases such ascocoa butter, glycerinated gelatin, hydrogenated vegetable oils,mixtures of polyethylene glycols of various molecular weights and fattyacid esters of polyethylene glycol.

When a compound according to this invention is administered into a humansubject, the daily dosage will normally be determined by the prescribingphysician with the dosage generally varying according to the age,weight, sex and response of the individual patient, as well as theseverity of the patient's symptoms.

In one exemplary application, a suitable amount of compound isadministered to a mammal undergoing treatment for cancer. Administrationoccurs in an amount between about 0.1 mg/kg of body weight to about 60mg/kg of body weight per day, preferably of between 0.5 mg/kg of bodyweight to about 40 mg/kg of body weight per day.

The instant compounds are also useful in combination with knowntherapeutic agents and anti-cancer agents. For example, instantcompounds are useful in combination with known anti-cancer agents.Combinations of the presently disclosed compounds with other anti-canceror chemotherapeutic agents are within the scope of the invention.Examples of such agents can be found in Cancer Principles and Practiceof Oncology by V. T. Devita and S. Hellman (editors), 6^(th) edition(Feb. 15, 2001), Lippincott Williams & Wilkins Publishers. A person ofordinary skill in the art would be able to discern which combinations ofagents would be useful based on the particular characteristics of thedrugs and the cancer involved. Such anti-cancer agents include, but arenot limited to, the following: estrogen receptor modulators, androgenreceptor modulators, retinoid receptor modulators, cytotoxic/cytostaticagents, antiproliferative agents, prenyl-protein transferase inhibitors,HMG-CoA reductase inhibitors and other angiogenesis inhibitors,inhibitors of cell proliferation and survival signaling, apoptosisinducing agents and agents that interfere with cell cycle checkpoints.The instant compounds are particularly useful when co-administered withradiation therapy.

In an embodiment, the instant compounds are also useful in combinationwith known anti-cancer agents including the following: estrogen receptormodulators, androgen receptor modulators, retinoid receptor modulators,cytotoxic agents, antiproliferative agents, prenyl-protein transferaseinhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors,reverse transcriptase inhibitors, and other angiogenesis inhibitors.

“Estrogen receptor modulators” refers to compounds that interfere withor inhibit the binding of estrogen to the receptor, regardless ofmechanism. Examples of estrogen receptor modulators include, but are notlimited to, tamoxifen, raloxifene, idoxifene, LY353381, LY117081,toremifene, fulvestrant,4-[7-2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-1-piperidinyl)ethoxy]phenyl}-2H-1-benzopyran-3-yl]-phenyl-2,2-imethylpropanoate,4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

“Androgen receptor modulators” refers to compounds which interfere orinhibit the binding of androgens to the receptor, regardless ofmechanism. Examples of androgen receptor modulators include finasterideand other 5α-reductase inhibitors, nilutamide, flutamide, bicalutamide,liarozole, and abiraterone acetate.

“Retioid receptor modulators” refers to compounds which interfere orinhibit the binding of retinoids to the receptor, regardless ofmechanism. Examples of such retinoid receptor modulators includebexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid,α-difluoromethylornithine, ILX23-7553,trans-N-4′-hydroxyphenyl)retinamide, and N-4-carboxyphenyl retinamide.“Cytotoxic/cytostatic agents” refer to compounds which cause cell deathor inhibit cell proliferation primarily by interfering directly with thecell's functioning or inhibit or interfere with cell mytosis, includingalkylating agents, tumor necrosis factors, intercalators, hypoxiaactivatable compounds, microtubule inhibitors/rnicrotubule-stabilizingagents, inhibitors of mitotic kinesins, inhibitors of kinases involvedin mitotic progression, antimetabolites; biological response modifiers;hormonal/anti-hormonal therapeutic agents, haematopoietic growthfactors, monoclonal antibody targeted therapeutic agents, topoisomeraseinhibitors, proteasome inhibitors and ubiquitin ligase inhibitors.

Examples of cytotoxic agents include, but are not limited to, sertenef,cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine,prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin,oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfantosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa,lobaplatin, satraplatin, profiromycin, cisplatin, irofulven,dexifosfamide, cis-aminedichloro(2-methyl-pyridine)platinum,benzylguanine, glufosfamide, GPX100, (trans, trans,trans)-bis-mu-hexane-1,6-diamine)-mu-[diamine-platinum(lI)]bis[diamine(chloro)platinum(II)]tetrachloride, diarizidinylspermine, arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin,pinafide, valrubicin, amrubicin, antineoplaston,3′-deamino-3′-morpholino-13-deoxo-10-hydroxycarminomycin, annamycin,galarubicin, elinafide, MEN10755, and4-demethoxy-3-deamino-3-aziridinyl4-methylsulphonyl-daunorubicin (see WO00/50032).

An example of a hypoxia activatable compound is tirapazamine.

Examples of proteasome inhibitors include but are not limited tolactacystin and bortezomib.

Examples of microtubule inhibitors/microtubule-stabilising agentsinclude paclitaxel, vindesine sulfate,3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine, docetaxol, rhizoxin,dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881,BMS184476, vinflunine, cryptophycin,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide,anhydrovinblastine,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide,TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and6,288,237) and BMS188797.

Some examples of topoisomerase inhibitors are topotecan, hycaptamine,irinotecan, rubitecan,6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin,9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-6H)propanamine,1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]-indolizino[1,2b]quinoline-10,13(9H,15H)dione,lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350,BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane,2′-diethylamino-2′-deoxy-etoposide, GL331,N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide,asulacrine, (5a, 5aB,8aa,9b)-9-[2-[N-[2-dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydro0xy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3′,4′:6,7)naphtho(2,3-d)-1,3-dioxol-6-one,2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium,6,9-bis[(2-aminoethyl)amino]benzo[g]isoguinoline-5,10-dione,5-(3-aminopropylamino)-7,10-dihydroxy-2-2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one,N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide,N-(2-(dimethylamino)ethyl)acridine-4-carboxamide,6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one, dimesna.

Examples of inhibitors of mitotic kinesins, and in particular the humanmitotic kinesin KSP, are described in PCT Publications WO 01/30768 andWO 01/98278, WO 03/050,064 (Jun. 19, 2003), WO 03/050,122 (Jun. 19,2003), WO 03/049,527 (Jun. 19, 2003), WO 03/049,679 (Jun. 19, 2003), WO03/049,678 (Jun. 19, 2003) and WO 03/39460 (May 15, 2003) and pendingPCT Appl. Nos. US03/06403 (filed Mar. 4, 2003), US03/15861 (filed May19, 2003), US03/15810 (filed May 19, 2003), US03/18482 (filed Jun. 12,2003) and US03/18694 (filed Jun. 12, 2003). In an embodiment inhibitorsof mitotic kinesins include, but are not limited to inhibitors of KSP,inhibitors of MKLP1, inhibitors of CENP-E, inhibitors of MCAK,inhibitors of Kif14, inhibitors of Mphosph1 and inhibitors of Rab6-KIFL.

“Inhibitors of linases involved in mitotic progression” include, but arenot limited to, inhibitors of aurora kinase, inhibitors of Polo-likekinases (PLK) (in particular inhibitors of PLK-1), inhibitors of bub-1and inhibitors of bub-R1.

“Antiproliferative agents” includes antisense RNA and DNAoligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001,and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin,doxifluridine, trinetrexate, fludarabine, capecitabine, galocitabine,cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed,paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed,nelzarabine, 2′-deoxy-2′-methylidenecytidine,2′-fluoromethylene-2′-deoxycytidine,N-[5-2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl)urea,N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno-heptopyranosyl]adenine,aplidine, ecteinascidin, troxacitabine,4-[2-aminooxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-yl-(S)-ethyl]-2,5-thienoyl-L-glutarnicacid, aminopterin, 5-flurouracil, alanosine,11-acetyl-8-(carbamoyloxymethyl)-4formyl-6-methoxy-14-oxa-1,11-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-ylacetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase,2′-cyano-2′-deoxy-N4-palmitoyl-1-B-D-arabino furanosyl cytosine and3-aminopyridine-2-carboxaldehyde thiosemicarbazone.

Examples of monoclonal antibody targeted therapeutic agents includethose therapeutic agents which have cytotoxic agents or radioisotopesattached to a cancer cell specific or target cell specific monoclonalantibody. Examples include Bexxar.

“HMG-CoA reductase inhibitors” refers to inhibitors of3-hydroxy-3-methylglutaryl-CoA reductase. Compounds which haveinhibitory activity for HMG-CoA reductase can be readily identified byusing assays well-known in the art. For example, see the assaysdescribed or cited in U.S. Pat. No. 4,231,938 at col. 6, and WO 84/02131at pp.30-33. The terms “HMG-CoA reductase inhibitor” and “inhibitor ofHMG-CoA reductase” have the same meaning when used herein.

Examples of HMG-CoA reductase inhibitors that may be used include butare not limited to lovastatin (MEVACOR®; see U.S. Pat. Nos. 4,231,938,4,294,926 and 4,319,039), simvastatin (ZOCOR®; see U.S. Pat. Nos.4,444,784, 4,820,850 and 4,916,239), pravastatin (PRAVACHOL®; see U.S.Pat. Nos. 4,346,227, 4,537,859, 4,410,629, 5,030,447 and 5,180,589),fluvastatin (LESCOL®; see U.S. Pat. Nos. 5,354,772, 4,911,165,4,929,437, 5,189,164, 5,118,853, 5,290,946 and 5,356,896) andatorvastatin (LIPITOR®; see U.S. Pat. Nos. 5,273,995, 4,681,893,5,489,691 and 5,342,952). The structural formulas of these andadditional HMG-CoA reductase inhibitors that may be used in the instantmethods are described at page 87 of M. Yalpani, “Cholesterol LoweringDrugs”, Chemistry & Industry, pp. 85-89 (5 Feb. 1996) and U.S. Pat. Nos.4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitor as usedherein includes all pharmaceutically acceptable lactone and open-acidforms (i.e., where the lactone ring is opened to form the free acid) aswell as salt and ester forms of compounds which have HMG-CoA reductaseinhibitory activity, and therefor the use of such salts, esters,open-acid and lactone forms is included within the scope of thisinvention. An illustration of the lactone portion and its correspondingopen-acid form is shown below as structures I and II.

In HMG-CoA reductase inhibitors where an open-acid form can exist, saltand ester forms may be formed from the open-acid, and all such forms areincluded within the meaning of the term “HMG-CoA reductase inhibitor” asused herein. In an embodiment, the HMG-oA reductase inhibitor isselected from lovastatin and simvastatin, and in a further embodiment,simvastatin. Herein, the term “pharmaceutically acceptable salts” withrespect to the HMG-CoA reductase inhibitor shall mean non-toxic salts ofthe compounds employed in this invention which are generally prepared byreacting the free acid with a suitable organic or inorganic base,particularly those formed from cations such as sodium, potassium,aluminum, calcium, lithium, magnesium, zinc and tetramethylammonium, aswell as those salts formed from amines such as ammonia, ethylenediamine,N-methylglucamine, lysine, arginine, ornithine, choline,N,N′-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine,N-benzylphenethylamine,1-p-chlorobenzyl-2-pyrrolidine-1′-yl-methylbenz-imidazole, diethylamine,piperazine, and tris(hydroxymethyl)aminomethane. Further examples ofsalt forms of HMG-CoA reductase inhibitors may include, but are notlimited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate,bitartrate, borate, bromide, calcium edetate, camsylate, carbonate,chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate,estolate, esylate, fumarate, gluceptate, gluconate, glutamate,glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynapthoate, iodide, isothionate, lactate,lactobionate, laurate, malate, maleate, mandelate, mesylate,methylsulfate, mucate, napsylate, nitrate, oleate, oxalate, pamaote,palmitate, panthothenate, phosphate/diphosphate, polygalacturonate,salicylate, stearate, subacetate, succinate, tannate, tartrate,teoclate, tosylate, triethiodide, and valerate.

Ester derivatives of the described HMG-CoA reductase inhibitor compoundsmay act as prodrugs which, when absorbed into the bloodstream of awarm-blooded animal, may cleave in such a manner as to release the drugform and permit the drug to afford improved therapeutic efficacy.

“Prenyl-protein transferase inhibitor” refers to a compound whichinhibits any one or any combination of the prenyl-protein transferaseenzymes, including farnesyl-protein transferase (FPTase),geranylgeranyl-protein transferase type I (GGPTase-I), andgeranylgeranyl-protein transferase type-II (GGPTase-II, also called RabGGPTase). Examples of prenyl-protein transferase inhibiting compoundsinclude(±)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-3-chlorophenyl)-1-methyl-2(1H)-uinolinone,(−)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-uinolinone,(+)-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone,5(S)-n-butyl-1-(2,3-dimethylphenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone,(S)-1-(3-chlorophenyl)-4-[1-4-cyanobenzyl)-5-imidazolylmethyl]-5-[2-(ethanesulfonyl)methyl)-2-piperazinone,5(S)-n-Butyl-1-(2-methylphenyl)4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone,1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-2-methyl-5-imidazolylmethyl]-2-piperazinone,1-(2,2-diphenylethyl)-3-[N-1-(4-cyanobenzyl)-1H-imidazol-5-ylethyl)carbamoyl]piperidine,4-{5-[4-hydroxymethyl-4-(4-chloropyridin-2-ylmethyl)-piperidine-1-ylmethyl]-2-methylnimdazol-1-ylmethyl}benzonitrile,4-{5-[4-hydroxymethyl-4-3-chlorobenzyl)-piperidine-1-ylmethyl]-2-methylimidazol-1-ylmethyl}benzonitrile,4-{3-[4-(2-oxo-2H-pyridin-1-yl)benzyl]-3H-imidazol-4-ylmethyl}benzonitrile,4-{3-[4-(5chloro-2-oxo-2H-[1,2′]bipyridin-5′-ylmethyl]-3H-imidazol-4-ylmethyl}benzonitrile,4-{3-[4-(2-oxo-2H-[1,2′]bipyridin-5′-ylmethyl]-3H-imidazolylmethyl}benzonitrile,4-[3-(2-oxo-1-phenyl-1,2-dihydropyridin-4-ylmethyl)-3H-imidazol-4-ylmethyl}benzonitrile,18,19dihydro-19-oxo-5H,17H-6,10:12,16-dimetheno-1H-imidazo[4,3-c][1,11,4]dioxaazacyclo-nonadecine-9-carbonitrile,(±)-19,20-diydro-19-oxo-5H-18,21-ethano-12,14-etheno-6,10-metheno-22H-benzo[d]imidazo[4,3-k][1,6,9,12]oxatriaza-cyclooctadecine-9-carbonitrile,19,20-ihydro-19-oxo-5H,17H-18,21-ethano-6,10:12,16-dimetheno-22H-imidazo[3,4-h][1,8,11,14]oxatriazacycloeicosine-9-carbonitrile,and(±)-19,20-dihydro-3-methyl-19-oxo-5H-18,21-ethano-12,14-etheno-6,10-metheno-22H-benzo[d]imidazo[4,3-k][1,6,9,12]oxa-triazacyclooctadecine-9-carbonitrile.

Other examples of prenyl-protein transferase inhibitors can be found inthe following publications and patents: WO 96/30343, WO 97/18813, WO97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/29119, WO95/32987, U.S. Pat. No. 5,420,245, U.S. Pat. No. 5,523,430, U.S. Pat.No. 5,532,359, U.S. Pat. No. 5,510,510, U.S. Pat. No. 5,589,485, U.S.Pat. No. 5,602,098, European Patent Publ. 0 618 221, European PatentPubl. 0 675 112, European Patent Publ. 0 604 181, European Patent Publ.0 696 593, WO 94/19357, WO 95/08542, WO 95/11917, WO 95/12612, WO95/12572, WO 95/10514, U.S. Pat. No. 5,661,152, WO 95/10515, WO95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO 96/05529, WO96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO96/00736, U.S. Pat. No. 5,571,792, WO 96/17861, WO 96/33159, WO96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO 96/30362, WO96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO 96/31501, WO97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO 97/02920, WO97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO98/02436, and U.S. Pat. No. 5,532,359.

For an example of the role of a prenyl-protein transferase inhibitor onangiogenesis see European J. of Cancer, Vol. 35, No. 9, pp.1394-1401(1999).

“Angiogenesis inhibitors” refers to compounds that inhibit the formationof new blood vessels, regardless of mechanisnl Examples of angiogenesisinhibitors include, but are not limited to, tyrosine kinase inhibitors,such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) andElk-1/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived,or platelet derived growth factors, MMP (matrix metalloprotease)inhibitors, integrin blockers, interferon-α, interleukin-12, pentosanpolysulfate, cyclooxygenase inhibitors, including nonsteroidalanti-inflarinatories (NSAIDs) like aspirin and ibuprofen as well asselective cyclooxy-genase-2 inhibitors like celecoxib and rofecoxib(PNAS, Vol. 89, p. 7384 (1992); JNCI Vol. 69, p. 475 (1982); Arch.Opthalmol., Vol. 108 p.573 (1990); Anat. Rec., Vol. 238, p. 68 (1994);FEBS Letters, Vol. 372, p. 83 (1995); Clin, Orthop. Vol. 313, p. 76(1995); J. Mol. Endocrinol., Vol. 16, p.107 (1996); Jpn. J. Pharmacol.,Vol. 75, p. 105 (1997); Cancer Res., Vol. 57, p. 1625 (1997); Cell, Vol.93, p. 705 (1998); Intl. J. Mol. Med., Vol. 2, p. 715 (1998); J. Biol.Chem., Vol. 274, p. 9116 (1999)), steroidal anti-inflammatories (such ascorticosteroids, mineralocorticoids, dexamethasone, prednisone,prednisolone, methylpred, betamethasone), carboxyamidotriazole,combretastatin A4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol,thalidornide, angiostatin, troponin-1, angiotensin II antagonists (seeFernandez et al., J. Lab. Clin. Med. 105:141-145 (1985)), and antibodiesto VEGF (see, Nature Biotechnology, Vol. 17, pp.963-968 (October 1999);Kim et al., Nature, 362, 841-844 (1993); WO 00/44777; and WO 00/61186).

Other therapeutic agents that modulate or inhibit angiogenesis and mayalso be used in combination with the compounds of the instant inventioninclude agents that modulate or inhibit the coagulation and fibrinolysissystems (see review in Clin. Chen La. Med. 38:679-692 (2000)). Examplesof such agents that modulate or inhibit the coagulation and fibrinolysispathways include, but are not limited to, heparin (see Thromb. Haemost.80:10-23 (1998)), low molecular weight heparins and carboxypeptidase Uinhibitors (also known as inhibitors of active thrombin activatablefibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354(2001)). TAFIa inhibitors have been described in PCT Publication WO03/013,526 and U.S. Ser. No. 60/349,925 (filed Jan. 18, 2002).

“Agents that interfere with cell cycle checkpoints” refer to compoundsthat inhibit protein kinases that transduce cell cycle checkpointsignals, thereby sensitizing the cancer cell to DNA damaging agents.Such agents include inhibitors of ATR, ATM, the Chk1 and Chk2 kinasesand cdk and cdc kinase inhibitors and are specifically exemplified by7-hydroxystaurosporin, flavopiridol, CYC₂₀₂ (Cyclacel) and BMS-387032.

“Inhibitors of cell proliferation and survival signaling pathway” referto pharmaceutical agents that inhibit cell surface receptors and signaltransduction cascades downstream of those surface receptors. Such agentsinclude inhibitors of inhibitors of EGFR (for example gefitinib anderlotinib), inhibitors of ERB-2 (for example trastuzumab), inhibitors ofIGFR, inhibitors of cytokine receptors, inhibitors of MET, inhibitors ofPI3K (for example LY294002), serine/threonine kinases (including but notlimited to inhibitors of Akt such as described in WO 021083064, WO02/083139, WO 02/083140 and WO 021083138), inhibitors of Raf kinase (forexample BAY-43-9006 ), inhibitors of MEK (for example CI-1040 andPD-098059) and inhibitors of mTOR (for example Wyeth CCI-779). Suchagents include small molecule inhibitor compounds and antibodyantagonists.

“Apoptosis inducing agents” include activators of TNF receptor familymembers (including the TRAIL receptors).

The combinations with NSAID's are directed to the use of NSAID's whichare potent COX-2 inhibiting agents. For purposes of this specificationan NSAID is potent if it possesses an IC₅₀ for the inhibition of COX-2of 1 μM or less as measured by cell or microsomal assays.

The invention also encompasses combinations with NSAID's which areselective COX-2 inhibitors. For purposes of this specification NSAID'swhich are selective inhibitors of COX-2 are defined as those whichpossess a specificity for inhibiting COX-2 over COX-1 of at least 100fold as measured by the ratio of IC₅₀ for COX-2 over IC₅₀ for COX-1evaluated by cell or microsomal assays. Such compounds include, but arenot limited to those disclosed in U.S. Pat. No. 5,474,995, issued Dec.12, 1995, U.S. Pat. No. 5,861,419, issued Jan. 19, 1999, U.S. Pat. No.6,001,843, issued Dec. 14, 1999, U.S. Pat. No. 6,020,343, issued Feb. 1,2000, U.S. Pat. No. 5,409,944, issued Apr. 25, 1995, U.S. Pat. No.5,436,265, issued Jul. 25, 1995, U.S. Pat. No. 5,536,752, issued Jul.16, 1996, U.S. Pat. No. 5,550,142, issued Aug. 27, 1996, U.S. Pat. No.5,604,260, issued Feb. 18, 1997, U.S. Pat. No. 5,698,584, issued Dec.16, 1997, U.S. Pat. No. 5,710,140, issued Jan. 20, 1998, WO 94/15932,published Jul. 21, 1994, U.S. Pat. No. 5,344,991, issued Jun. 6, 1994,U.S. Pat. No. 5,134,142, issued Jul. 28, 1992, U.S. Pat. No. 5,380,738,issued Jan. 10, 1995, U.S. Pat. No. 5,393,790, issued Feb. 20, 1995,U.S. Pat. No. 5,466,823, issued November 14, 1995, U.S. Pat. No.5,633,272, issued May 27, 1997, and U.S. Pat. No. 5,932,598, issued Aug.3, 1999, all of which are hereby incorporated by reference.

Inhibitors of COX-2 that are particularly useful in the instant methodof treatment are:

-   3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone; and-   5-chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridine;-   or a pharmaceutically acceptable salt thereof.

General and specific synthetic procedures for the preparation of theCOX-2 inhibitor compounds described above are found in U.S. Pat. No.5,474,995, issued Dec. 12, 1995, U.S. Pat. No. 5,861,419, issued Jan.19, 1999, and U.S. Pat. No. 6,001,843, issued Dec. 14, 1999, all ofwhich are herein incorporated by reference.

Compounds that have been described as specific inhibitors of COX-2 andare therefore useful in the present invention include, but are notlimited to, the following:

-   or a pharmaceutically acceptable salt thereof.

Compounds which are described as specific inhibitors of COX-2 and aretherefore useful in the present invention, and methods of synthesisthereof, can be found in the following patents, pending applications andpublications, which are herein incorporated by reference: WO 94/15932,published Jul. 21, 1994, U.S. Pat. No. 5,344,991, issued Jun. 6, 1994,U.S. Pat. No. 5,134,142, issued Jul. 28, 1992, U.S. Pat. No. 5,380,738,issued Jan. 10, 1995, U.S. Pat. No. 5,393,790, issued Feb. 20, 1995,U.S. Pat. No. 5,466,823, issued November 14, 1995, U.S. Pat. No.5,633,272, issued May 27, 1997, and U.S. Pat. No. 5,932,598, issued Aug.3, 1999.

Compounds which are specific inhibitors of COX-2 and are thereforeuseful in the present invention, and methods of synthesis thereof, canbe found in the following patents, pending applications andpublications, which are herein incorporated by reference: U.S. Pat. No.5,474,995, issued Dec. 12, 1995, U.S. Pat. No. 5,861,419, issued Jan.19, 1999, U.S. Pat. No. 6,001,843, issued Dec. 14, 1999, U.S. Pat. No.6,020,343, issued Feb. 1, 2000, U.S. Pat. No. 5,409,944, issued Apr. 25,1995, U.S. Pat. No. 5,436,265, issued Jul. 25, 1995, U.S. Pat. No.5,536,752, issued Jul. 16, 1996, U.S. Pat. No. 5,550,142, issued Aug.27, 1996, U.S. Pat. No. 5,604,260, issued Feb. 18, 1997, U.S. Pat. No.5,698,584, issued Dec. 16, 1997, and U.S. Pat. No. 5,710,140, issuedJan. 20, 1998.

Other examples of angiogenesis inhibitors include, but are not limitedto, endostatin, ukrain, ranpirnase, IM862,5-methoxy4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate,acetyldinanaline,5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide,CM101,squalamine, combretastatin, RPI4610, NX31838, sulfated mannopentaosephosphate,7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl4,2-pyrrole]-carbonylimino]-bis-(1,3-naphthalenedisulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone(SU5416).

As used above, “integrin blockers” refers to compounds which selectivelyantagonize, inhibit or counteract binding of a physiological ligand tothe α_(v)β₃ integrin, to compounds which selectively antagonize, inhibitor counteract binding of a physiological ligand to the α_(v)β₅ integrin,to compounds which antagonize, inhibit or counteract binding of aphysiological ligand to both the α_(v)β₃ integrin and the α_(v)β₅integrin, and to compounds which antagonize, inhibit or counteract theactivity of the particular integrn(s) expressed on capillary endothelialcells. The term also refers to antagonists of the α_(v)β_(6,) α_(v)β₈,α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins. The term also refers toantagonists of any combination of α_(v)β₃, α_(v)β₅, α_(v)β₆, α_(v)β₈,α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins.

Some specific examples of tyrosine kinase inhibitors includeN-trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide,3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indolin-2-one,17-(allylamino)-17-demethoxygeldanamycin,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline,N-3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinanine, BIBX1382,2,3,9,10,11,12-hexahydro-10-hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one,SH268, genistein, STI571, CEP2563, 4-(3-chlorophenylamino)-5,6dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethane sulfonate,4-(3-bromro4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline,4-(4′-hydroxyphenyl)amino6,7-dimethoxyquinazoline, SU6668, ST1571A,N-4-chlorophenyl-4-4-pyridylmethyl)-1-phthalazinamine, and EMD121974.

Combinations with compounds other than anti-cancer compounds are alsoencompassed in the instant methods. For example, combinations of theinstantly claimed compounds with PPAR-γ (i.e., PPAR-gamma) agonists andPPAR-δ (i.e., PPAR-delta) agonists are useful in the treatment ofcertain malingnancies. PPAR-γ and PPAR-δ are the nuclear peroxisomeproliferator-activated receptors γ and δ. The expression of PPAR-γ onendothelial cells and its involvement in angiogenesis has been reportedin the literature (see J. Cardiovasc. Pharmacol. 1998; 31:909-913; J.Biol. Chem. 1999;274:9116-9121; Invest. Ophthalmol Vis. Sci. 2000;41:2309-2317). More recently, PPAR-γ agonists have been shown to inhibitthe angiogenic response to VEGF in vitro; both troglitazone androsiglitazone maleate inhibit the development of retinalneovascularization in mice. (Arch. Ophthamol. 2001; 119:709-717).Examples of PPAR-γ agonists and PPAR-γ/α agonists include, but are notlimited to, thiazolidinediones (such as DRF2725, CS-011, troglitazone,rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate,GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544,NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRF552926,2-[(5,7-ipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropionicacid (disclosed in U.S. Ser. No. 09/782,856), and2(R)-7-3-2-chloro-4-fluorophenoxy)phenoxy)propoxy)-2-ethylchromane-2-carboxylicacid (disclosed in U.S. Ser. Nos. 60/235,708 and 60/244,697).

Another embodiment of the instant invention is the use of the presentlydisclosed compounds in combination with gene therapy for the treatmentof cancer. For an overview of genetic strategies to treating cancer seeHall et al (Am J Hum Genet 61:785-789, 1997) and Kufe et al (CancerMedicine, 5th Ed, pp 876-889, B C Decker, Hamilton 2000). Gene therapycan be used to deliver any tumor suppressing gene. Examples of suchgenes include, but are not limited to, p53, which can be delivered viarecombinant virus-mediated gene transfer (see U.S. Pat. No. 6,069,134,for example), a uPA/uPAR antagonist (“Adenovirus-Mediated Delivery of auPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth andDissemination in Mice,” Gene Therapy, August 1998;5(8):1105-13), andinterferon gamma (J Immunol 2000; 164:217-222).

The compounds of the instant invention may also be administered incombination with an inhibitor of inherent multidrug resistance (MDR), inparticular MDR associated with high levels of expression of transporterproteins. Such MDR inhibitors include inhibitors of p-glycoprotein(P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833(valspodar).

A compound of the present invention may be employed in conjunction withanti-emetic agents to treat nausea or emesis, including acute, delayed,late-phase, and anticipatory emesis, which may result from the use of acompound of the present invention, alone or with radiation therapy. Forthe prevention or treatment of emesis, a compound of the presentinvention may be used in conjunction with other anti-emetic agents,especially neurokiini-1 receptor antagonists, 5HT3 receptor antagonists,such as ondansetron, granisetron, tropisetron, and zatisetron, GABABreceptor agonists, such as baclofen, a corticosteroid such as Decadron(dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten orothers such as disclosed in U.S. Pat. Nos. 2,789,118, 2,990,401,3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326 and 3,749,712, anantidoparninergic, such as the phenothiazines (for exampleprochlorperazine, fluphenaiine, thioridazine and mesoridazine),metoclopramide or dronabinol. For the treatment or prevention of emesisthat may result upon administration of the instant compounds,conjunctive therapy with an anti-emesis agent selected from aneurokinin-1 receptor antagonist, a 5HT3 receptor antagonist and acorticosteroid is preferred.

Neuroknin-1 receptor antagonists of use in conjunction with thecompounds of the present invention are fully described, for example, inU.S. Pat. Nos. 5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595,5,459,270, 5,494,926, 5,496,833, 5,637,699, 5,719,147; European PatentPublication Nos. EP 0 360 390, 0 394 989, 0 428 434, 0 429 366, 0 430771, 0 436 334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901, 0512 902, 0 514 273, 0 514 274, 0 514 275, 0 514 276, 0 515 681, 0 517589, 0 520 555, 0 522 808, 0 528 495, 0 532 456, 0 533 280, 0 536 817, 0545 478, 0 558 156, 0 577 394, 0 585 913,0 590 152, 0 599 538, 0 610793, 0 634 402, 0 686 629, 0 693 489, 0 694 535, 0 699 655, 0 699 674, 0707 006, 0 708 101, 0 709 375, 0 709 376, 0 714 891, 0 723 959, 0 733632 and 0 776 893; PCT International Patent Publication Nos. WO90/05525, 90/05729, 91/09844, 91/18899, 92/01688, 92/06079, 92/12151,92/15585, 92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93/00330,93/00331, 93/01159, 93/01165, 93/01169, 93/01170, 93/06099, 93/09116,93/10073, 93/14084, 93/14113, 93/18023, 93/19064, 93/21155, 93/21181,93/23380, 93/24465, 94/00440, 94/01402, 94/02461, 94/02595, 94/03429,94/03445, 94/04494, 94/04496, 94/05625, 94/07843, 94/08997, 94/10165,94/10167, 94/10168, 94/10170, 94/11368, 94/13639, 94/13663, 94/14767,94/15903, 94/19320, 94/19323, 94/20500, 94/26735, 94/26740, 94/29309,95/02595, 95/04040, 95/04042, 95/06645, 95/07886, 95/07908, 95/08549,95/11880, 95/14017, 95/15311, 95/16679, 95/17382, 95/18124, 95/18129,95/19344, 95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418,95/30674, 95/30687, 95/33744, 96/05181, 96/05193, 96/05203, 96/06094,96/07649, 96/10562, 96/16939, 96/18643, 96/20197, 96121661, 96/29304,96/29317, 96/29326, 96/29328, 96/31214, 96/32385, 96/37489, 97/01553,97/01554, 97/03066, 97/08144, 97/14671, 97/17362, 97/18206, 97/19084,97/19942 and 97/21702; and in British Patent Publication Nos. 2 266 529,2 268 931, 2 269 170, 2 269 590, 2 271 774, 2 292 144, 2 293 168, 2 293169, and 2 302 689. The preparation of such compounds is fully describedin the aforementioned patents and publications, which are incorporatedherein by reference.

In an embodiment, the neurokinin-1 receptor antagonist for use inconjunction with the compounds of the present invention is selectedfrom:2-(R)-1-(R)-(3,5-bis(triluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)morpholine,or a pharmaceutically acceptable salt thereof, which is described inU.S. Pat. No. 5,719,147.

A compound of the instant invention may also be administered with anagent useful in the treatment of anernia. Such an anemia treatment agentis, for example, a continuous eythropoiesis receptor activator (such asepoetin alfa).

A compound of the instant invention may also be administered with anagent useful in the treatment of neutropenia Such a neutropeniatreatment agent is, for example, a hematopoietic growth factor whichregulates the production and function of neutrophils such as a humangranulocyte colony stimulating factor, (G-CSF). Examples of a G-CSFinclude filgrastiu.

A compound of the instant invention may also be administered with anim-munologic-enhancing drug, such as levarnisole, isoprinosine andZadaxin.

Thus, the scope of the instant invention encompasses the use of theinstantly claimed compounds in combination with a second compoundselected from: 1) an estrogen receptor modulator, 2) an androgenreceptor modulator, 3) retinoid receptor modulator, 4) acytotoxic/cytostatic agent, 5) an antiproliferative agent, 6) aprenyl-protein transferase inhibitor, 7) an HMG-CoA reductase inhibitor,8) an HIV protease inhibitor, 9) a reverse transcriptase inhibitor, 10)an angiogenesis inhibitor, 11) a PPAR-γ agonists, 12) a PPAR-δ agonists,13) an inhibitor of inherent multidrug resistance, 14) an anti-emeticagent, 15) an agent useful in the treatment of anemia, 16) an agentuseful in the treatment of neutropenia, 17) an immunologic-enhancingdrug, 18) an inhibitor of cell proliferation and survival signaling, 19)an agent that interfers with a cell cycle checkpoint; and 20) anapoptosis inducing agent.

The term “administration” and variants thereof (e.g., “administering” acompound) in reference to a compound of the invention means introducingthe compound or a prodrug of the compound into the system of the animalin need of treatment. When a compound of the invention or prodrugthereof is provided in combination with one or more other active agents(e.g., a cytotoxic agent, etc.), “administration” and its variants areeach understood to include concurrent and sequential introduction of thecompound or prodrug thereof and other agents.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombination of the specified ingredients in the specified amounts.

The term “therapeutically effective amount” as used herein means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue, system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician.

The term “treating cancer” or “treatment of cancer” refers toadministration to a mammal afflicted with a cancerous condition andrefers to an effect that alleviates the cancerous condition by killingthe cancerous cells, but also to an effect that results in theinhibition of growth and/or metastasis of the cancer.

In an embodiment, the angiogenesis inhibitor to be used as the secondcompound is selected from a tyrosine linase inhibitor, an inhibitor ofepidermal-derived growth factor, an inhibitor of fibroblast-derivedgrowth factor, an inhibitor of platelet derived growth factor, an P(matrix metalloprotease) inhibitor, an integrin blocker, interferon-αinterleukin-12, pentosan polysulfate, a cyclooxygenase inhibitor,carboxyamidotriazole, combretastatin A-4, squalamine,6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin,troponin-1, or an antibody to VEGF. In an embodiment, the estrogenreceptor modulator is tamoxifen or raloxifene.

Also included in the scope of the claims is a method of treating cancerthat comprises administering a therapeutically effective amount of acompound of Formula I in combination with radiation therapy and/or incombination with a compound selected from: 1) an estrogen receptormodulator, 2) an androgen receptor modulator, 3) retinoid receptormodulator, 4) a cytotoxic/cytostatic agent, 5) an antiproliferativeagent, 6) a prenyl-protein transferase inhibitor, 7) an HMG-CoAreductase inhibitor, 8) an HIV protease inhibitor, 9) a reversetranscriptase inhibitor, 10) an angiogenesis inhibitor, 11) a PPAR-γagonists, 12) a PPAR-δ agonists, 13) an inhibitor of inherent multidrugresistance, 14) an anti-emetic agent, 15) an agent useful in thetreatment of anemia, 16) an agent useful in the treatment ofneutropenia, 17) an immmunologic-enhancing drug, 18) an inhibitor ofcell proliferation and survival signaling, 19) an agent that interferswith a cell cycle checkpoint; and 20) an apoptosis inducing agent.

And yet another embodiment of the invention is a method of treatingcancer that comprises administering a therapeutically effective amountof a compound of Formula I in combination with paclitaxel ortrastuzumab.

The invention ftrther encompasses a method of treating or preventingcancer that comprises administering a therapeutically effective amountof a compound of Formula I in combination with a COX-2 inhibitor.

The instant invention also includes a pharmaceutical composition usefulfor treating or preventing cancer that comprises a therapeuticallyeffective amount of a compound of Formula I and a compound selectedfrom: 1) an estrogen receptor modulator, 2) an androgen receptormodulator, 3) a retinoid receptor modulator, 4) a cytotoxic/cytostaticagent, 5) an antiproliferative agent, 6) a prenyl-protein transferaseinhibitor, 7) an HMG-CoA reductase inhibitor, 8) an HIV proteaseinhibitor, 9) a reverse transcriptase inhibitor, 10) an angiogenesisinhibitor, 11) a PPAR-γ agonist, 12) a PPAR-δ agonists; 13) an inhibitorof cell proliferation and survival signaling, 14) an agent thatinterfers with a cell cycle checkpoint; and an apoptosis inducing agent.

The invention further comprises the use of the instant compounds in amethod to screen for other compounds that bind to KSP. To employ thecompounds of the invention in a method of screening for compounds thatbind to KSP kinesin, the KSP is bound to a support, and a compound ofthe invention (which is a mitotic agent) is added to the assay.Alternatively, the compound of the invention is bound to the support andKSP is added. Classes of compounds among which novel binding agents maybe sought include specific antibodies, non-natural binding agentsidentified in screens of chemical libraries, peptide analogs, etc. Ofparticular interest are screening assays for candidate agents that havea low toxicity for human cells. A wide variety of assays may be used forthis purpose, including labeled in vitro protein-protein binding assays,electrophoretic mobility shift assays, immunoassays for protein binding,functional assays (phosphorylation assays, etc.) and the like.

The determination of the binding of the mitotic agent to KSP may be donein a number of ways. In a preferred embodiment, the mitotic agent (thecompound of the invention) is labeled, for example, with a fluorescentor radioactive moiety and binding determined directly. For example, thismay be done by attaching all or a portion of KSP to a solid support,adding a labeled mitotic agent (for example a compound of the inventionin which at least one atom has been replaced by a detectable isotope),washing off excess reagent, and determining whether the amount of thelabel is that present on the solid support Various blocking and washingsteps may be utilized as is known in the art.

By “labeled” herein is meant that the compound is either directly orindirectly labeled with a label which provides a detectable signal,e.g., radioisotope, fluorescent tag, enzyme, antibodies, particles suchas magnetic particles, chemiluminescent tag, or specific bindingmolecules, etc. Specific binding molecules include pairs, such as biotinand streptavidin, digoxin and antidigoxin etc. For the specific-bindingmembers, the complementary member would normally be labeled with amolecule which provides for detection, in accordance with knownprocedures, as outlined above. The label can directly or indirectlyprovide a detectable signal.

In some embodiments, only one of the components is labeled. For example,the kinesin proteins may be labeled at tyrosine positions using ¹²⁵I, orwith fluorophores. Alternatively, more than one component may be labeledwith different labels; using ¹²⁵I for the proteins, for example, and afluorophor for the mitotic agents.

The compounds of the invention may also be used as competitors to screenfor additional drug candidates. “Candidate bioactive agent” or “drugcandidate” or grammatical equivalents as used herein describe anymolecule, e.g., protein, oligopeptide, small organic molecule,polysaccharide, polynucleotide, etc., to be tested for bioactivity. Theymay be capable of directly or indirectly altering the cellularproliferation phenotype or the expression of a cellular proliferationsequence, including both nucleic acid sequences and protein sequences.In other cases, alteration of cellular proliferation protein bindingand/or activity is screened. Screens of this sort may be performedeither in the presence or absence of microtubules. In the case whereprotein binding or activity is screened, preferred embodiments excludemolecules already known to bind to that particular protein, for example,polymer structures such as microtubules, and energy sources such as ATP.Preferred embodiments of assays herein include candidate agents which donot bind the cellular proliferation protein in its endogenous nativestate termed herein as “exogenous” agents. In another preferredembodiment, exogenous agents further exclude antibodies to KSP.

Candidate agents can encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 100 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding andlipophilic binding, and typically include at least an amine, carbonyl,hydroxyl, ether, or carboxyl group, preferably at least two of thefunctional chemical groups. The candidate agents often comprise cyclicalcarbon or heterocyclic structures and/or aromatic or polyaromaticstructures substituted with one or more of the above functional groups.Candidate agents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof. Particularly preferred arepeptides.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

Competitive screening assays may be done by combining KSP and a drugcandidate in a first sample. A second sample comprises a mitotic agent,KSP and a drug candidate. This may be performed in either the presenceor absence of microtubules. The binding of the drug candidate isdetermined for both samples, and a change, or difference in bindingbetween the two samples indicates the presence of an agent capable ofbinding to KSP and potentially modulating its activity. That is, if thebinding of the drug candidate is different in the second sample relativeto the first sample, the drug candidate is capable of binding to KSP.

In a preferred embodiment, the binding of the candidate agent isdetermined through the use of competitive binding assays. In thisembodiment, the competitor is a binding moiety known to bind to KSP,such as an antibody, peptide, binding partner, ligand, etc. Undercertain circumstances, there may be competitive binding as between thecandidate agent and the binding moiety, with the binding moietydisplacing the candidate agent.

In one embodiment, the candidate agent is labeled. Either the candidateagent, or the competitor, or both, is added first to KSP for a timesufficient to allow binding, if present Incubations may be performed atany temperature which facilitates optimal activity, typically betweenabout 4 and about 40° C.

Incubation periods are selected for optimum activity, but may also beoptimized to facilitate rapid high throughput screening. Typicallybetween 0.1 and 1 hour will be sufficient Excess reagent is generallyremoved or washed away. The second component is then added, and thepresence or absence of the labeled component is followed, to indicatebinding.

In a preferred embodiment, the competitor is added first, followed bythe candidate agent. Displacement of the competitor is an indication thecandidate agent is binding to KSP and thus is capable of binding to, andpotentially modulating, the activity of KSP. In this embodiment, eithercomponent can be labeled. Thus, for example, if the competitor islabeled, the presence of label in the wash solution indicatesdisplacement by the agent. Alternatively, if the candidate agent islabeled, the presence of the label on the support indicatesdisplacement.

In an alternative embodiment, the candidate agent is added first, withincubation and washing, followed by the competitor. The absence ofbinding by the competitor may indicate the candidate agent is bound toKSP with a higher affinity. Thus, if the candidate agent is labeled, thepresence of the label on the support, coupled with a lack of competitorbinding, may indicate the candidate agent is capable of binding to KSP.

It may be of value to identify the binding site of KSP. This can be donein a variety of ways. In one embodiment, once KSP has been identified asbinding to the mitotic agent, KSP is fragmented or modified and theassays repeated to identify the necessary components for binding.

Modulation is tested by screening for candidate agents capable ofmodulating the activity of KSP comprising the steps of combining acandidate agent with KSP, as above, and determining an alteration in thebiological activity of KSP. Thus, in this embodiment, the candidateagent should both bind to KSP (although this may not be necessary), andalter its biological or biochemical activity as defined herein. Themethods include both in vitro screening methods and in vivo screening ofcells for alterations in cell cycle distribution, cell viability, or forthe presence, morphology, activity, distribution, or amount of mitoticspindles, as are generally outlined above.

Alternatively, differential screening may be used to identify drugcandidates that bind to the native KSP, but cannot bind to modified KSP.

Positive controls and negative controls may be used in the assays.Preferably all control and test samples are performed in at leasttriplicate to obtain statistically significant results. Incubation ofall samples is for a time sufficient for the binding of the agent to theprotein. Following incubation, all samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples may be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g., albumin,detergents, etc which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

These and other aspects of the invention will be apparent from theteachings contained herein.

Assays

The compounds of the instant invention described in the Examples weretested by the assays described below and were found to have kinaseinhibitory activity. Other assays are known in the literature and couldbe readily performed by those of skill in the art (see, for example, PCTPublication WO 01/30768, May 3, 2001, pages 18-22).

-   I. Kinesin ATPase In Vitro Assay-   Cloning and Expression of Human Poly-Histidine Tagged KSP Motor    Domain (KSP(367H))

Plasmids for the expression of the human KSP motor domain construct werecloned by PCR using a pBluescript full length human KSP construct(Blangy et al., Cell, vol.83, pp1159-1169, 1995) as a template. TheN-terminal primer 5′-GCAACGATTAATATGGCGTCGCAGCCAAATTCGTCTGCGAAG(SEQ.ID.NO.: 1) and the C-terminal primer 5′-GCAACGCRCGAGTCAGTGATGATGGTGGTGATGCTGATTCACTTCAGGCTTATTCAATAT (SEQ.ID.NO.: 2) were used toamplify the motor domain and the neck linker region. The PCR productswere digested with AseI and XhoI, ligated into the NdeIIXhoI digestionproduct of pRSETa (Invitrogen) and transformed into E. coli BL21 (DE3).

Cells were grown at 37° C. to an OD₆₀₀ of 0.5. After cooling the cultureto room temperature expression of KSP was induced with 100 μM IPTG andincubation was continued overnight Cells were pelleted by centrifugationand washed once with ice-cold PBS. Pellets were flash-frozen and stored−80° C.

-   Protein Purification

Cell pellets were thawed on ice and resuspended in lysis buffer (50 mMK-HEPES, pH 8.0, 250 mM KCl, 0.1% Tween, 10 mM imidazole, 0.5 mM Mg-ATP,1 mM PMSF, 2 mM benzimidine, 1× complete protease inhibitor cocktail(Roche)). Cell suspensions were incubated with 1 mg/ml lysozyme and 5 mMβ-mercaptoethanol on ice for 10 minutes, followed by sonication (3×30sec). All subsequent procedures were performed at 4° C. Lysates werecentrifuged at 40,000×g for 40 minutes. Supernatants were diluted andloaded onto an SP Sepharose column (Pharmacia, 5 ml cartridge) in bufferA (50 mM K-HEPES, pH 6.8, 1 mM MgCl₂, 1 mM EGTA, 10 μM Mg-ATP, 1 mM DTT)and eluted with a 0 to 750 mM KCl gradient in buffer A. Fractionscontaining KSP were pooled and incubated with Ni—NTA resin (Qiagen) forone hour. The resin was washed three times with buffer B (Lysis bufferminus PMSF and protease inhibitor cocktail), followed by three 15-minuteincubations and washes with buffer B. Finally, the resin was incubatedand washed for 15 minutes three times with buffer C (same as buffer Bexcept for pH 6.0) and poured into a colurmn. KSP was eluted withelution buffer (identical to buffer B except for 150 mM KCl and 250 mMimidazole). KSP-containing fractions were pooled, made 10% in sucrose,and stored at −80° C.

Microtubules are prepared from tubulin isolated from bovine brain.Purified tubulin (>97% MAP-free) at 1 mg/ml is polymerized at 37° C. inthe presence of 10 μM paclitaxel, 1 mM DTT, 1 mM GTP in BRB80 buffer (80mM K-PIPES, 1 mM EGTA, 1 mM MgCl₂ at pH 6.8). The resulting microtubulesare separated from non-polymerized tubulin by ultracentrifugation andremoval of the supernatant. The pellet, containing the microtubules, isgently resuspended in 10 μM paclitaxel, 1 mM DTT, 50 μg/ml ampicillin,and 5 μg/ml chloramphenicol in BRB80.

The kinesin motor domain is incubated with microtubules, 1 MM ATP (1:1MgCl₂: Na-ATP), and compound at 23° C. in buffer containing 80 mMK-HEPES (pH 7.0), 1 mM EGTA, 1 mM DTT, 1 mM MgCl₂, and 50 mM KCl. Thereaction is terminated by a 2-10 fold dilution with a final buffercomposition of 80 mM HEPES and 50 mM EDTA. Free phosphate from the ATPhydrolysis reaction is measured via a quinaldine red/ammonium molybdateassay by adding 150 μl of quench C buffer containing a 2:1 ratio ofquench A:quench B. Quench A contains 0.1 mg/ml quinaldine red and 0.14%polyvinyl alcohol; quench B contains 12.3 mM ammonium molybdatetetrahydrate in 1.15 M suffuric acid. The reaction is incubated for 10minutes at 23° C., and the absorbance of the phosphomolybdate complex ismeasured at 540 nm.

The compounds of the instant invention may be tested in the above assayand an IC₅₀ found.

-   II. Cell Proliferation Assay

Cells are plated in 96-well tissue culture dishes at densities thatallow for logarithmic growth over the course of 24, 48, and 72 hours andallowed to adhere overnight. The following day, compounds are added in a10-point, one-half log titration to all plates. Each titration series isperformed in triplicate, and a constant DMSO concentration of 0.1% ismaintained throughout the assay. Controls of 0.1% DMSO alone are alsoincluded. Each compound dilution series is made in media without sermThe final concentration of serum in the assay is 5% in a 200 μL volumeof media Twenty microliters of Alamar blue staining reagent is added toeach sample and control well on the titration plate at 24, 48, or 72hours following the addition of drug and returned to incubation at 37°C. Alamar blue fluorescence is analyzed 6-12 hours later on a CytoFluorII plate reader using 530-560 nanometer wavelength excitation, 590nanometer emission.

A cytotoxic EC₅₀ is derived by plotting compound concentration on thex-axis and average percent inhibition of cell growth for each titrationpoint on the y-axis. Growth of cells in control wells that have beentreated with vehicle alone is defined as 100% growth for the assay, andthe growth of cells treated with compounds is compared to this value.Proprietary in-house software is used to calculate percent cytotoxicityvalues and inflection points using logistic 4-parameter curve fitting.Percent cytotoxicity is defined as:% cytotoxicity:(Fluorescence_(control))−(Flourescence_(sample))×100×(Fluorescence_(control))⁻¹

-   The inflection point is reported as the cytotoxic EC₅₀.-   III. Evaluation of Mitotic Arrest and Apoptosis by FACS

FACS analysis is used to evaluate the ability of a compound to arrestcells in mitosis and to induce apoptosis by measuring DNA content in atreated population of cells. Cells are seeded at a density of 1.4×10⁶cells per 6 cm² tissue culture dish and allowed to adhere overnight.Cells are then treated with vehicle (0.1% DMSO) or a titration series ofcompound for 8-16 hours. Following treatment, cells are harvested bytrypsinization at the indicated times and pelleted by centrifugation.Cell pellets are rinsed in PBS and fixed in 70% ethanol and stored at 4°C. overnight or longer.

For FACS analysis, at least 500,000 fixed cells are pelleted and the 70%ethanol is removed by aspiration. Cells are then incubated for 30 min at4° C. with RNase A (50 Kunitz units/ml) and propidium iodide (50 μg/ml),and analyzed using a Becton Dickinson FACSCaliber. Data (from 10,000cells) is analyzed using the Modfit cell cycle analysis modelingsoftware (Verity Inc.).

An EC₅₀ for mitotic arrest is derived by plotting compound concentrationon the x-axis and percentage of cells in the G2/M phase of the cellcycle for each titration point (as measured by propidium iodidefluorescence) on the y-axis. Data analysis is performed using theSigmaPlot program to calculate an inflection point using logistic4-parameter curve fitting. The inflection point is reported as the EC₅₀for mitotic arrest. A similar method is used to determine the compoundEC₅₀ for apoptosis. Here, the percentage of apoptotic cells at eachtitration point (as determined by propidium iodide fluorescence) isplotted on the y-axis, and a similar analysis is carried out asdescribed above.

-   IV. Immunofluorescence Microscopy to Detect Monopolar Spindles

Methods for immunofluorescence staining of DNA, tubulin, and pericentrinare essentially as described in Kapoor et al. (2000) J. Cell Biol. 150:975-988. For cell culture studies, cells are plated on tissue culturetreated glass chamber slides and allowed to adhere overnight. Cells arethen incubated with the compound of interest for 4 to 16 hours. Afterincubation is complete, media and drug are aspirated and the chamber andgasket are removed from the glass slide. Cells are then permeabiued,fixed, washed, and blocked for nonspecific antibody binding according tothe referenced protocol. Paraffin-enbedded tumor sections aredeparaffinized with xylene and rehydrated through an ethanol seriesprior to blocking. Slides are incubated in primary antibodies (mousemonoclonal anti-α-tubulin antibody, clone DM1A from Sigma diluted 1:500;rabbit polyclonal anti-pericentrin antibody from Covance, diluted1:2000) overnight at 4° C. After washing, slides are incubated withconjugated secondary antibodies (FTC-conjugated donkey anti-mouse IgGfor tubulin; Texas red-conjugated donkey anti-rabbit IgG forpericentrin) diluted to 15 μg/ml for one hour at room temperature.Slides are then washed and counterstained with Hoechst 33342 tovisualize DNA. Immunostained samples are imaged with a 100× oilimmersion objective on a Nikon epifluorescence microscope usingMetamorph deconvolution and imaging software.

EXAMPLES

Examples provided are intended to assist in a further understanding ofthe invention. Particular materials employed, species and conditions areintended to be illustrative of the invention and not limiting of thereasonable scope thereof.

Step 1: 4-Allyl-4-phenyl-1,3-oxazolidin-2-one (1-4)

To a suspension of 15.8 g (416 mmol) of LAH powder in 600 mL of diethylether was added 18.3 g (90 mmol) of α-allyl-α-phenylglycine ethyl ester(-3) (prepared according to: Van Betsbrugge et. al. Tetrahtedron, 1997,53, 9233-9240) in 75 mL of diethyl ether at such a rate as to maintaingentle reflux. After stirring overnight at room temperature, thereaction was carefully quenched with 27 mL of water, followed by 27 mLof 15% NaOH and finally 82 mL of water. A quantity of Na₂SO₄ was added,and the mixture was stirred for 1 h. The solids were then filtered offand the solution concentrated. The residue was dissolved in 300 mL ofCH₂Cl₂, dried over Na₂SO₄, and concentrated to provide the amino alcoholas a colorless oil. The amino alcohol (4.5 g, 25 mmol) was dissolved in50 mL of CH₂Cl₂ and cooled to 0° C. Following the addition of 5.4 mL (53mmol) of triethylamine and 4.5 g (28 mmol) of 1,1′-carbonyldiinidazole,the mixture was warmed to room temperature and allowed to stir for 4 h.The reaction was then dumped into a separatory fimnel, washed twice with1M HCl, water, dried over Na₂SO₄, and concentrated to obtainoxazolidinone 1-4 as a colorless oil. Data for 1-4: 1HNMR (500 MHz,CDCl₃) δ 7.4 -7.2 (m, 5H), 6.6 (s, 1H), 5.6 -5.5 (m, 1H), 5.2 (m, 2H),4.5 (d, 1H), 4.35 (d, 1H), 2.8 (m, 1H), 2.6 (m, 1H) ppm.

Step 2: Diester (1-5)

A solution of 68 g (334.6 mmol) of 1-4 in 500 mL of CH₂Cl₂ was cooled to−78° C. and ozone was bubbled through the solution until a pale bluecolor persisted. O₂ was then bubbled through the solution for 15minutes, followed by 30 minutes with N₂. At that time, 491 mL (6.7moles) of dimethyl sulfide was added, and the solution was stirredovernight while slowly coming to room temperature. The volitiles wereremoved by rotary evaporation to provide a brown oil. This material wassuspended in 1L of tBuOH, and 200 mL (1.9 moles) of 2-methyl-2-butenewas added. To this solution was then added dropwise a mixture of 160 g(1.33 moles) of NaH₂PO₄ and 70 g (774 mmol) of NaClO₂ in 800 mL of H₂O.After the addition was complete, the mixture was stirred for anadditional 4 h. After separating the layers, the organic wasconcentrated by rotary evaporation, the residue was dissolved in EtOAcand placed in a separatory funnel with the aqueous phase from thereaction. After separation, the aqueous phase was extracted 3× withEtOAc, dried over Na₂SO₄, and concentrated to provide ˜90 g of a yellowgum. This residue was suspended in 500 mL of MeOH, and HCl gas wasbubbled through the solution until it was nearly refluxing. The flaskwas then capped and allowed to stir overnight while cooling to roomtemperature. The volitiles were removed by rotary evaporation, theresidue was loaded onto a silica gel column in CH₂Cl₂, and eluted withEtOAc/hexanes to provide the methyl ester as a pale orange gum. Thisresidue was dissolved in 500 mL of THF, cooled to 0° C., and 32.6 mL(220.5 mmol) of tert-butyl bromoacetate was added, followed by 10.6 g ofNaH (264.6 mmol of a 60% suspension). After the mixture was allowed towarm to room temperature and stir overnight, it was quenched with asaturated NH₄Cl solution, and extracted twice with EtOAc. The combinedorganic layers were then washed with brine, dried over Na₂SO₄,concentrated, and the residue purified by silica gel chromatography withEtOAc/hexanes to provide 1-5 as a thick pale yellow gum. Data for 1-5:¹HNMR (500 MHz, CDCl₃) δ 7.4 -7.3 (m, 5H), 4.65 (d, 1H), 4.55 (d, 1H),3.9 (d, 1H), 3.65 (s, 1H), 3.5 (d, 1H), 3.35 (d, 1H), 3.2 (d, 1H), 1.4(s, 9H) ppm. HRMS (ES) calc'd M+Na for C₁₈H₂₃NO₆: 372.1423. Found:372.1412.

Step 3: 7a-Phenyldihydro-1H-pyrrolo[1,2-c][1,3]oxazole-3,6(5H)-dione(1-6)

To a solution of 18.6 g (53 mmol) of 1-5 in 150 mL of TBF at −78° C. wasadded dropwise 58.6 mL (58.6 mmol) of a 1M solution of LiHMDS in THF.After stirring for 1 h at that temperature, the cooling bath was removedand the reaction was allowed to warm to room temperature and stirovernight. The mixture was quenched with a saturated NH₄Cl solution,extracted twice with EtOAc, washed twice with brine, dried over Na₂SO₄and concentrated. The residue was dissolved in 60 mL of formic acid andheated at 100° C. for 24 h. The volatiles were removed under vacuum andthe residue was triturated with CH₂Cl₂/hexanes/Et₂O to provide 1-6 as abeige solid. Data for 1-6: ¹HNMR (500 MHz, CDCl₃) δ 7.5-7.3 (m, 5H), 4.7(d, 1H), 4.3 (d, 1H), 4.2 (d, 1H), 3.5 (d, 1H), 3.1 (d, 1H), 2.95 (d,1H), 2.9 (d, 1H) ppm.

Step 4:6-(2.5-Difluorophenyl)-7a-phenyl-5,7a-dihydro-1H-pyrrolo[1,2-c][1,3]oxazol-3-one(1-7)

To a suspension of 2.2 g (10 mmol) of 1-7 in 150 mL of THF at −78° C.was added dropwise 12.2 mL (12.2 mmol) of a 1M solution of NaHMDS inTHF. After stirring for 30 min, the solution was allowed to warm to 0°C. and held there for 1 h. The solution was then cooled back down to−78° C. and a solution of 4.35 g (12.2 mmol) ofN-phenylbis(trifluoromethanesulphonimide) in 10 mL of THF was added. Thecooling bath was removed and the mixture was allowed to warm to roomtemperature and stir overnight. The mixture was quenched with asaturated NH₄Cl solution, extracted twice with EtOAc, washed twice withbrine, dried over Na₂SO₄ and concentrated. The residue was dissolved in75 mL of DME and 18 mL of water. To this mixture was added 1.29g (30mmol) of LiCl, 3.2 g (30 mmol) of Na₂CO₃, and 4.8 g (30 mmol) of2,5-difluorophenylboronic acid. The solution was then degassed with N₂for 1 minute, followed by the addition of 630 mg (0.5 mmol) oftetrakis(triphenylphosphine)palladium (0). The reaction was heated at90° C. for 3 h, cooled to room temperature, diluted with saturatedNaHCO₃, and extracted twice with EtOAc. The combined organic layers werewashed with brine, dried over Na₂SO₄, concentrated, and the residuepurified by silica gel chromatography with CH₂Cl₂/hexanes to provide 1-7as a white solid. Data for 1-7: ¹HNMR (500 MHz, CDCl₃) δ 7.5-7.3 (m,5H), 7.1-6.9 (m, 3H), 6.8 (s, 1H), 4.9 (d, 1H), 4.75 (d, 1H), 4.5 (d,1H), 4.23 (d, 1H) ppm. HRMS (ES) calc'd M+H for C₁₈H₁₃F₂NO₂: 314.0987.Found: 314.0993.

Step 5:2-({[tert-Butyl(dimethyl)silyl]oxy}methyl)-4-(2,5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pvrrole(1-8)

A suspension of 1.75 g (5.6 mmol) 1-7 in 15 mL of EtOH and 10 mL of 3 MNaOH was heated at 60° C. for 3 h, cooled to room temperature and dumpedinto a separatory funnel with EtOAc and brine. The layers wereseparated, the aqueous phase was extracted twice with EtOAc, thecombined organic phases were washed twice with brine, dried over Na₂SO₄,and concentrated to provide a white solid. To this flask was added 30 mLof CH₂Cl₂, 1.5 g (22.3 mmol) of imidazole and 1.76 g (11.7 mmol) ofTBSCl, and the resultant suspension was stirred overnight. The reactionwas diluted with CH₂Cl₂, washed twice with water, dried over Na₂SO₄,concentrated, and the residue purified by silica gel chromatography withEtOAc/hexanes to provide 1-8 as a white solid. Data for 1-8: ¹HNMR (500MHz, CDCl₃) δ 7.6-7.3 (m, 5H), 7.1-6.9 (m, 3H), 6.75 (s, 1H), 4.25 (d,1H), 4.1 (d, 1H), 3.95 (d, 1H), 3.75 (d, 1H), 0.9 (s, 9H), 0.1 (s, 3H),0.05 (s, 3H) ppm.

Step 6: Enantiomeric Resolution of Intermediate 1-8

Resolution of the enantiomers was carried out chromatographically usinga Chiralpak AD® 10×50 cm column with 1% isopropanol in hexanes (with0.1% diethylamine) at 150 mL/min. Analytical HPLC analysis of the eluenton a 4×250 mm Chiralpak AD® column with 1% isopropanol in hexanes (with0.1% diethylamine) at 1 mL/min indicated that first eluting, activeenantiomer has R_(t)=5.5 min and the second enantiomer has R_(t)=6.9min.

Step 7: Carbamoyl chloride 1-9

To a solution of 1.95 g (6.6 mmol) of triphosgene in 25 mL of TBF at 0°C. was added a solution of 1.31 g (3.3 mmol) of the first elutingenantiomer of 1-8 and 915 μL (6.6 mmol) of triethylamine in 10 mL ofTHF. The ice bath was removed and the reaction was allowed to warm toroom temperature and stir for 3 h. The reaction was then partitionedbetween water and EtOAc, the layers were separated, dried over Na₂SO₄,and concentrated to provide 1-9 as a brown oil. Data for 1-9:

HRMS (ES) calc'd M+H for C₂₄H₂₈ClF₂NO₂Si: 464.1619. Found: 464.1625.

-   Alternate Synthesis to Diester 1-5

To a biphasic mixture of 14.8 g (73 mmol) of 1-4 and 110 mL of CH₂Cl₂,110 mL of CH₃CN, and 320 mL of water was added approximately 200 mg ofruthenium(III) chloride hydrate. Sodium periodate (85.6 g, 400 mmol) wasthen added portion-wise over 1 h with rapid stirring. After the additionwas complete, the reaction was allowed to stir for 4 h more at roomtemperature. The mixture was diluted with 500 mL of water and 1.5 L ofEtOAc, and the solids were removed by filtration. The filtrate wasplaced in a separatory finnel, the phases separated, the aqueous phaseextracted twice with EtOAc, the combined organic phases washed twicewith brine, and dried over Na₂SO₄. Following concentration, the darkbrown solid was dissolved in 250 mL of MeOH and HCl(g) was slowly passedthrough the solution at a rate so as not to increase the temperature ofthe solution above 35° C. After 5 min, the reaction was capped andallowed to stir at room temperature overnight. The volatiles were thenremoved on a rotary evaporator, and the residue was purified by silicagel chromatography with EtOAc/hexanes to provide 13.6 g (58 mmol) of themethyl ester as a viscous oil. This residue was then dissolved in 200 mLof ThF, cooled to 0° C., and 10.3 mL (70 mmol) of tert-butylbromoacetate was added, followed by 2.8 g of NaH (70 mmol of a 60%suspension). After the mixture was allowed to warm to room temperatureand stir overnight, it was quenched with a saturated NH₄Cl solution, andextracted twice with EtOAc. The combined organic layers were then washedwith brine, dried over Na₂SO₄, concentrated, and the residue purified bysilica gel chromatography with EtOAc/hexanes to provide 1-5 as acolorless oil. Data for 1-5: ¹HNMR (500 MHz, CDCl₃) δ 7.4-7.3 (m, 5H),4.65 (d, 1H), 4.55 (d, 1H), 3.9 (d, 1H), 3.65 (s, 3H), 3.5 (d, 1H), 3.35(d, 1H), 3.2 (d, 1H), 1.4 (s, 9H) ppm. HRMS (ES) calc'd M+Na forC₁₈H23NO₆: 372.1423. Found: 372.1412.

Step 1: Methyl 4-methylene-2-phenylprolinate (1B-2)

An aqueous solution (300 mL) of phenyl glycine methyl ester-HCl (100 g)was neutralized to pH 8 with 10N NaOH. The aqueous solution wasextracted with EtOAc (3×200 mL). The combined organic extracts weredried over MgSO₄, filtered, and concentrated. The residue (56.7 g, 344mmol) was dissolved in trimethylorthoformate (100 mL) and treated withbenzaldehyde (34.9 mL, 36.4 g, 344 mmol). After stirring for 2 h, thereaction was diluted with Et₂O (200 mL) and washed with water (3×50 mL).The organic solution was dried over MgSO₄, filtered, and concentrated. Aportion of the imine residue (26.8 g, 100 mmol) was dissolved indichloromethane (240 mL) and treated with 160 mL of 10N NaOH, methallyldichloride (50.0 g, 400 mmol), and Bu₄NHSO₄ (3.59 g). After stirring for10 h at rt, the reaction was diluted with dichloromethane and theorganic solution separated, dried over MgSO₄, filtered, andconcentrated. The residue was redissolved in Et₂O/1N HCl (200 mL/200 mL)and stirred for 2 h. The aqueous phase was separated and neutralizedwith 10N NaOH (to pH 8). The aqueous mixture was extracted with EtOAc(3×200 mL). The combined organic solutions were dried over MgSO₄,filtered and concentrated. The residue was dissolved in water andneutralized (to pH 8). Extraction of this mixture with EtOAc (×3)followed by drying over MgSO₄, filtratration, and concentration providedcrude 1B-2. Purification of this residue by flash chromatography (SiO₂;30% EtOAc/hexanes) provided pure 1B-2.

Data for 1B-2: ¹HNMR (500 MHz, CDCl₃) δ 7.51 (m, 2H), 7.42 (m, 3H), 5.03(s, 1H), 4.95 (s, 1H), 3.71 (m, 5H), 3.41 (rn, 1H), 2.80 (m, 1H) ppm.

Step 2: 7a-Phenyldihydro-1H-pyrrolo[1,2-c][1.3]oxazole-3,6(5H)-dione(1-6)

A suspension of LiAlH₄ (7.14 g, 188 mmol) in THF (500 mL) was cooled to0° C. and treated with a solution of ester 1B-2 (10.2 g, 47 mmol) in THF(50 mL) over 20 min. After stirring for 30 min at 0° C., the reactionwas cautiously quenched by the addition of water (7.1 mL), 15% aq NaOH(7.1 mL), and H₂O (21.3 mL). Solid Na₂SO₄ was added and the mixturestirred for 40 min. The mixture was filtered and concentrated. Theresidue (8.2 g, 43.3 mmol) was dissolved in dichloromethane (300 mL) andtreated with triethylamine (9.0 mL, 6.5 g, 65.0 mmol) andcarbonyldiimidazole (9.14 g, 56.4 mmol). After stirring for 48 h at rt,the reaction was diluted with dichloromethane and washed with 1N HCl andbrine. The organic solution was concentrated and not further purified. Asolution of the residue 1B-3 (9.2 g, 42.8 mmol) in dichloromethane (200mL) was cooled to −78° C. and ozone was passed through the solutionuntil a blue color persisted. The solution was purged and treated withdimethylsulfide (35 mL). After gradual warming to rt overnight, thesolution was concentrated to a yellow solid. Trituration of this solidwith Et₂O provided pure 1-6. Data for 1-6: ¹HNMR (500 MHz, CDCl3) δ 87.5-7.3 (m, 5H), 4.7 (d, 1H), 4.3 (d, 1H), 4.2 (d, 1H), 3.5 (d, 1H), 3.1(d, 1H), 2.95 (d, 1H), 2.9 (d, 1H) ppm

The compounds of the instant invention are prepared by reacting compound1-9 with reagents (prepared as illustrated in Schemes B and D-F) asshown in Schemes C and G-O above.

Step 1: (±)-N-BOC-trans-3-fluoro-4(methylamnino)pyrrolidine 2-3

A cooled solution (−78 ° C.) of N-BOC-3-pyrrolidinone (1.0 g, 5.41 mmol)in THF (50 mL) was treated with a solution of NaHMDS (7.03 mL of a 1Msoln in THF). After stirring for 30 min, the solution was treated withchlorotriethylsilane (1.18 mL, 7.03 mmol). The reaction was stirred for1 hr at −78 ° C., warmed to 0 ° C., diluted with 1:1 brine/water andhexanes. The mixture was extracted with EtOAc. The organic solution wasdried over Na₂SO₄, filtered, and concentrated. The residue was notfurther purified. A solution of the 3-fluoro-N-BOC-pyrrolidinone (0.31g, 1.53 mmol) in dichloroethane (10 mL) was treated with methylarnine(1.53 mL of a 2M soln in THF) and Na(OAc)₃BH (0.49 g, 2.3 mmol). Afterstirring for 12 h at room temperature, the reaction was diluted withEtOAc and washed with satd NaHCO₃. The organic solution was dried overMgSO₄, filtered, and concentrated. The residue was purified by flashchromatography (SiO₂; 80% CHCl₃/10% MeOH/10% EtOAc) to provide racemic2-3. Data for 2-3: ¹HNMR (500 MHz, CDCl₃) δ 5.11 (br d, J=55 Hz, 1H),3.85-3.62 (m, 2H), 3.51 (m, 1H), 3.20 (m, 1H), 3.03 (m, 1H), 2.50 (s,3H), 1.46 (s, 9H) ppm.

Step 2:(2S)-4-2,5-Difluorophenyl)-N-[(3R,4R)-4-fluoropyrrolidin-3-yl]-2-hydroxymethyl)N-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide2-5a and(2S)-4-2,5-Difluorophenyl)-N-[(3S,4S)-4-fluoropyrrolidin-3-yl]-2-(hydroxymethyl)N-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide2-5b

A solution of the the carbamoyl chloride 1-9 (0.24 g, 0.52 mmol),fluoropyrrolidine 2-3 (0.114 g, 0.52 mmol), and iPr₂NEt (0.177 mL, 1.04mmol) in dioxane (5 mL) was heated at 90° C. for 12 h. The reaction wascooled to room temperature, diluted with EtOAc and washed with satd aqNH₄Cl and brine. The organic solution was dried over MgSO₄, filtered,and concentrated. The residue was purified by flash chromatography(SiO₂; 10% EtOAc/hexanes) to provide diastereomers 2-4a and 2-4b.Diastereomer 2-4a (0.1 g) was dissolved in dioxane (2 mL) and treatedwith HCl (2 mL of a 4M soln in dioxane). After stirring for 2 h, thereaction was neutized with satd aq NaHCO₃ and extracted with EtOAc. Theorganic solution was dried over MgSO₄, filtered, and concentrated. Flashchromatography on SiO₂ (95/2.5/2.5 dichloromethane/NH₄OH/MeOH) affordedpure first eluting 2-5a. 2-5b was prepared in a similar fashion.

-   Data for 2-5a: ¹HNMR (500 MHz, CDCl₃) δ 7.40-7.35 (m, 5H), 7.07-6.96    (m, 3H), 6.30 (s, 1H), 5.34-5.23 (br d, J=55 Hz, 1H), 4.86 (d, J=14    Hz, 1H), 4.75 (d, J=14 Hz, 1H), 4.52 (m, 1H), 4.05 (m, 2H), 3.27 (m,    3H1), 3.08 (s, 3H) ppm.-   Data for 2-5b: ¹HNMR (500 MHz, CDCl₃) δ 7.41-7.37 (m, 5H), 7.09-6.96    (m, 3H), 6.32 (s, 1H), 5.36-5.23 (dt, J=55, 4 Hz, 1H), 4.90 (d, J=15    Hz, 1H), 4.67 (d, J=15 Hz, 1H), 4.45 (d, J=12H, 1H), 4.03 (m, 2H),    3.29 (m, 3H), 3.18 (s, 3H) ppm.

Step 3:(2S)-4-(2,5-Difluorophenyl)-N-[(3R,4R)-4-fluoro-1-methylpyrrolidin-3-yl]-2-(hydroxymethyl)N-phenyl-2,5-dhydro-1H-pyrrole-1-carboxamide2-6a and(2S)-4-2,5-Difluorophenyl)-N-[(3S,4S)-4-fluoro-1-methylpyrrolidin-3-yl]-2-(hydroxymethyl)N-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide2-6b

A solution of amine 2-5a (0.050 g, 0.116 mmol), aqueous formaldehyde(0.030 mL, 0.348 mmol), NaCNBH₃ (0.024 g, 0.348 mmol), AcOH (10 μL) inMeOH (4.0 mL) was stirred at rt for 12 h. The reaction was treated withsatd aq NaHCO₃ (30 uL) and concentrated. The reaction was purified byflash chromatography (SiO₂; 95/2.5/2.5 dichloromethane/NH₄OH/MeOH) toprovide 2-6a. 2-6b was prepared in a similar fashion.

-   Data for 2-6a: ¹HNMR (500 MHz, CDCl₃) δ 7.41-7.36 (ma, 4H),    7.29-7.26 (m, 1H), 7.08-6.95 (m, 3H), 6.29 (s, 1H), 5.31-5.18 (br d,    J=55 Hz, 1H), 4.89-4.86 (d, J=14 Hz, 1H), 4.78-4.75 (d, J=14 Hz,    1H), 4.52 (d, J=12 Hz, 1H), 4.37-4.28 (m, 1H), 4.05 (d, J=12 Hz,    1H), 3.10 (s, 3H), 2.99-2.81 (m, 4H), 2.43 (s, 3H) ppm.-   Data for 2-6b: ¹HNMR (500 MHz, CDCl₃) δ 7.39-7.26 (m, 5H), 7.08-6.96    (m, 3H), 6.33 (s, 1H), 5.35-5.22 (dt, J=54, 4.5 Hz, 1H), 4.94-4.91    (d, J=14 Hz, 1H), 4.65-4.62 (d, J=14 Hz, 1H), 4.43-4.32 (m, 2H),    4.02-3.99 (d, J=13 Hz, 1H), 3.22 (s, 3H), 3.16-3.02 (m, 2H),    2.71-2.61 (m, 2H), 2.44 (s, 3H) ppm.

Step 1:(2S)-4-(2,5-Difluorophenyl)-N-[(3S,5S)-5-(fluoromethyl)-N-BOC-pyrrolidin-3-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide3-2

A solution of azide 3-1 [0.68 g, 2.78 mmol; prepared from (2S,4R)-4-hydroxy-L-proline ethyl ester according to Rosen, et al, J. Med.Chem. 1988, 31, 1598-1611] in THF (20 mL) was treated with polymer boundPh₃P (0.58 g, 4.0 mmol) and heated at 50° C. for 3 h. The reaction wascooled to rt and stirred for 48 h. The reaction was treated with water(20 mL) and warmed to 50° C. for 1 h. The reaction was cooled to roomtemperature, filtered through celite, and concentrated. A portion of theresidue (0.103 g, 0.474 mmol) was treated with carbamoyl chloride 1-9(0.22 g, 0.474 mmol), triethylamine (0.129 mL, 0.948 mmol), and acatalytic amount of DMAP. The reaction was stirred for 48 h at roomtemperature. The reaction was diluted with EtOAc and washed with 10%citric acid, satd aq NaHCO₃, and brine. The organic solution was driedover MgSO₄, filtered and concentrated. The residue was purified bychromatography on SiO₂ (gradient 100% hexanes to 50% EtOAc/hexanes over45 min) provided 3-2 as a white solid. LRMS M+H=646.4.

Step 2:(2S)-4-(2,5-Difluorophenyl)-N-[(3S,5S)-5-(fluoromethyl)-pyrrolidin-3-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide3-3

A solution of 3-2 (0.18 g, 0.279 mmol) in DMF (3 mL) at 0° C was treatedwith NaH (0.009 g, 0.362 mmol) and iodomethane (0.023 mL, 0.362 mol).The reaction was stirred for 12 h with gradual warming to rt A secondportion (0.3 eq) of both iodomethane and NaH were added. After stirringfor another 3 h, the reaction was diluted with EtOAc, washed with water,10% citric acid, satd aq NaHCO₃, and brine. The organic solution wasdried over MgSO₄, filtered and concentrated. The residue was purified bychromatography (SiO₂; 100% hexanes to 3/1 hexanes/EtOAc over 45 min).This material (0.150 g, 0.227 mmol) was dissolved in EtOAc (1.2 mL) andtreated with a solution of HCl in dioxane (4M, 1.2 mL). After stirringfor 2 h, the reaction was diluted with EtOAc and satd aq NaHCO₃ (˜3 mL)was added. The mixture was concentrated and rediluted with 15% Na₂CO₃.The mixture was extracted with 2/1 CHCl₃: EtOH (×3) and the combinedorganic solution was concentrated. The residue was diluted withdichloromethane washed with brine, dried over Na₂SO₄, filtered andconcentrated to provide 3-3 Data for 3-3: ¹HNMR (500 MHz, CD₃OD) δ7.38-7.31 (m, 4H), 7.25-7.22 (m, 2H), 7.19-7.14 (m, 1H), 7.10-7.06 (m,1H), 6.31 (s, 1H), 4.95 (d, J=14 Hz, 1H), 4.77 (d, J=14 Hz, 1H),4.61 (d,J=11.2 Hz, 1H), 4.51 (m, 1H), 4.43 (m, 1H), 4.34 (m, 1H), 4.20 (d, J=11Hz, 1H), 4.11 (m, 1H), 3.08 (m, 1H), 2.09 (m, 4H), 2.02 (m, 1H), 1.56(m, 1H) ppm.

Step 3:(2S)-4-2,5-Difluorophenyl)-N-[(3S,5S)-5-(fluoromethyl)-1-methylpyrroldin-3-yl]-2-(hydroxyethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide3-4

A solution of 3-3 (0.90 g, 0.202 mmol) in MeOH (2 mL) was treated withAcOH (20 uL), aqueous formaldehyde (0.018 g, 0.606 mmol), and NaCNBH₃(0.038 g 0.606 mmol). After stimng for 1 h at room temperature, thereaction was treated with satd aqueous NaHCO₃, and extracted with EtOAc.The combined organic solutions were washed with brine, dried over MgSO₄,filtered and concentrated. The residue was purified by chromatography(SiO₂, 100% EtOAc to 10% MeOH in EtOAc) to provide 34.

-   Data for 3-4: ¹HNMR (500 MHz, CDCl₃) δ 7.35 (m, 5H), 6.96 (m, 3H),    6.27 (s, 1H), 5.39 (m, 1H), 4.85 (d, J=15 Hz, 1H), 4.53 (m, 4H),    3.98(dd, J=12, 3.7 Hz, 1H), 3.14 (dd, J=11, 2.2 Hz, 1H), 3.07 (s,    3H), 2.53 (mn, 1H), 2.42 (m, 4H), 1.70 (m, 1H) ppm.

(2S)-4-(2,5-Difluorophenyl)-N-[(3S,5R)-5-(fluoromethyl)-1-methylpyrrolidin-3-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide3-5

Compound 3-5 was prepared by sequence analogous to Scheme 3 beginningwith (2R, 4R-)4 -hydroxy-D-proline methyl ester. Data for 3-5: ¹HNMR(500 MHz, CDCl₃) δ 7.37 (mn, 5H), 6.97 (m, 3H), 6.26 (s, 1H), 5.23 (d,J=5.5 Hz, 1H), 4.84 (d, J=14 Hz, 1H), 4.63 (d, J=14 Hz, 1H), 4.45 (m,2H), 4.37 (m, 2H), 4.01 (dd, J=11, 3 Hz, 1H), 3.24 (m, 1H), 2.93 (s,3H), 2.76 (m, 1H), 2.51 (m, 1H), 2.44 (s, 3H) ppm.

1. A compound of Formula I:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:a is 0 or 1; b is 0 or 1; m is 0, 1, or 2; n is 0, 1, 2 or 3; r is 0 or1; s is 0 or 1; t is 0, 1 or 2; R¹ and R² are independently selectedfrom: H, (C₁-C₆)alkyl, aryl, heterocyclyl and (C₃-C₆)cycloalkyl,optionally substituted with one, two or three substituents selected fromR⁷; R³ is selected from 1) hydrogen; 2) C₁-C₁₀ alkyl; 3) C₁-C₁₀alkyl-O—R^(d), 4) C₂-C₁₀ alkenyl-O—R^(d), 5) C₂-C₁₀ alkynyl-O—R^(d), 6)(C₁-C₆-alkylene)_(n)C₃-C₈ cycloalkyl-O—R^(d), 7) C₁-C₁₀alyl-(C═O)_(b)—NR^(c)R^(c)′, 8) C₂-C₁₀ alkenyl-C═O)_(b)NR^(c)R^(c)′, 9)C₂-C₁₀ anyl-(C═O)_(b)NR^(c)R^(c)′, 10) (C₁-C₆-alkyene)_(n)C₃-C₈cycloalkyl-C═O)_(b)NRCR^(c)′, 11) C₁-C₁₀ alkyl-S(O)_(m)—R^(d), 12) C₂C₁₀alkenyl-S(O)_(m)—R^(d), 13) C₂-C₁₀ alkynyl-S(O)_(m)—R^(d), 14)(C1-C₆-alkylene)_(n)C₃-C₈ cycloalkyl-S(O)_(m)—R^(d), said alkyl,alkenyl, alkynyl and cycloalkyl are optionally substituted with one ormore substituents selected from R⁶; R⁴ is independently selectedfrom: 1) (C═O)_(a)O_(b)C₁-C₁₀ alkyl, 2) (C═O)_(a)O_(b)aryl, 3) CO₂H, 4)halo, 5) CN, 6) OH, 7) O_(b)C₁-C₆ perfluoroalkyl, 8)O_(a)(C═O)_(b)NR⁸R⁹, 9) S(O)_(m)R^(a), 10) S(O)₂NR⁸R⁹, said ailcyl,aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl optionallysubstituted with one, two or three substituents selected from R⁷; R⁵ isselected from: 1) hydrogen; 2) (C═O)_(a)O_(b)C₁-C₁₀ alkyl, 3)(C═O)_(a)O_(b)aryl, 4) CO₂H, 5) halo, 6) CN, 7) OH, 8) O_(b)C₁-C₆perfluoroalkyl, 9) O_(a)(C═O)_(b)NR⁸R⁹, 10) S(O)_(m)R^(a), 11)S(O)₂NR⁸R⁹, said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, andcycloalkyl optionally substituted with one, two or three substituentsselected from R⁷; R⁶ is independently selected from: 1)(C═O)_(a)O_(b)C₁-C₁₀ alkyl, 2) (C═O)_(a)O_(b)aryl, 3) C₂-C₁₀ alkenyl, 4)C₂-C₁₀ alkynyl, 5) (C═O)_(a)O_(b) heterocyclyl, 6) CO₂H, 7) halo, 8) CN,9) OH, 10) O_(b)C₁-C₆ perfluoroalkyl, 11) O_(a)(C═O)_(b)NR⁸R⁹, 12)S(O)_(m)R^(a), 13) S(O)₂NR⁸R⁹, 14) oxo, 15) CHO, 16) (N═O)R⁸R⁹, or 17)(C═O)_(a)O_(b)C₃-C₈ cycloalkyl, said alkyl, aryl, alkenyl, alkynyl,heterocyclyl, and cycloalkyl optionally substituted with one, two orthree substituents selected from R⁷; R⁷ is selected from: 1)(C═O)_(r)OS(C₁-C₁₀)alkyl, 2) O_(r)(C₁-C₃)perfluoroalkyl, 3) oxo, 4) OH,5) halo, 6) CN, 7) (C₂-C₁₀)alkenyl, 8) (C₂-C₁₀)alkynyl, 9)(C═O)_(r)O_(s)(C₃-C₆)cycloalkyl, 10) (C═O)_(r)O_(s)(C₀-C₆)allylene-aryl,11) (C═O)_(r)O_(s)(C₀-C₆)alkylene-heterocyclyl, 12)(C═O)_(r)O_(s)(C₀-C₆)alkylene-N(R^(b))₂, 13) C(O)R^(a), 14)(C₀-C₆)alklene-CO₂R^(a), 15) C(O)H, 16) (C₀-C₆)alkylene-CO₂H, and 17)C(O)N(R^(b))₂, 18) S(O)_(m)R^(a), and 19) S(O)₂N(R^(b))₂; said alkyl,alkenyl, alkynyl, cycloalkyl, aryl, alkylene and heterocyclyl isoptionally substituted with up to three substituents selected fromR^(b), OH, (C₁-C₆)alkoxy, halogen, CO₂H, CN, O(C═O)C₁-C₆ alkyl, oxo, NO₂and N(R^(b))₂; R⁸ and R⁹ are independently selected from: 1) H, 2)(C═O)O_(b)C₁-C₁₀ alkyl, 3) (C═O)O_(b)C₃-C₈ cycloalkyl, 4)(C═O)O_(b)aryl, 5) (C═O)O_(b)heterocyclyl, 6) C₁-C₁₀ alkyl, 7) aryl, 8)C₂-C₁₀ alkenyl, 9) C₂-C₁₀ alkynyl, 10) heterocyclyl, 11) C₃-C₈cycloalkyl, 12) SO₂R^(a), and 13) (C═O)NR^(b) ₂, said alkyl, cycloalkyl,aryl, heterocylyl, alkenyl, and alkynyl is optionally substituted withone, two or three substituents selected from R⁷, or R⁸ and R⁹ can betaken together with the nitrogen to which they are attached to form amonocyclic or bicyclic heterocycle with 3-7 members in each ring andoptionally containing, in addition to the nitrogen, one or twoadditional heteroatoms selected from N, O and S, said monocyclic orbicyclic heterocycle optionally substituted with one, two or threesubstituents selected from R⁷; R¹⁰ is selected from: F and —CH₂F; R¹¹and R¹² are independently selected from: H and —CH₂F; R^(ox) is absentor is oxo; R^(a) is independently selected from: (C₁-C₆)alkyl,(C₃-C₆)cycloalkyl, aryl, or heterocyclyl, optionally substituted withone, two or three substituents selected from R⁷; R^(b) is independentlyselected from: H, (C₁-C₆)alkyl, aryl, heterocyclyl, (C₃-C₆)cycloalkyl,(C═O)OC₁-C₆ alkyl, (C═O)C₁-C₆ alkyl, (C═O)aryl, (C═O)heterocyclyl,(C═O)NR^(e)R^(e)′ or S(O)₂R^(a), optionally substituted with one, two orthree substituents selected from R⁷; R^(c) and R^(c)′ are independentlyselected from: H, (C₁-C₆)alkyl, aryl, NH₂OH, OR^(a), —(C₁-C₆)alkyl-OH,—(C₁-C₆)alkyl-O—(C₁-C₆)alkyl, (C═O)OC₁-C₆alkyl, (C═O)C₁-C₆ alkyl,(C═O)aryl, (C═O)heterocyclyl, (C═O)NR^(e)R^(e)′, S(O)₂R^(a) and—(C₁-C₆)alkyl-N(R^(b))₂, wherein the alkyl is optionally substitutedwith one, two or three substituents selected from R⁷; or R^(c) andR^(c)′ can be taken together with the nitrogen to which they areattached to form a monocyclic or bicyclic heterocycle with 3-7 membersin each ring and optionally containing, in addition to the nitrogen, oneor two additional heteroatoms selected from N, O and S, said monocyclicor bicyclic heterocycle optionally substituted with one, two or threesubstituents selected from R⁷; R^(d) is selected from: H, (C₁-C₆)alkyl,—C₂-C₆)alkyl-OH, —C₁-C₆)alkyl-O—C₁-C₆)alkyl and C₁-C₆)alkyl-N(R^(b))₂,wherein the alkyl is optionally substituted with one, two or threesubstituents selected from R⁷;; R^(e) and R^(e)′ are independentlyselected from H, (C₁-C₆)alkyl, aryl, heterocyclyl and (C₃-C₆)cycloalkyl,optionally substituted with one, two or three substituents selected fromR⁷; or R^(e) and R^(e)′ can be taken together with the nitrogen to whichthey are attached to form a monocyclic or bicyclic heterocycle with 3-7members in each ring and optionally containing, in addition to thenitrogen, one or two additional heteroatoms selected from N, O and S,said monocyclic or bicyclic heterocycle optionally substituted with one,two or three substituents selected from R⁷.
 2. The compound according toclaim 1 of Formula II:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:a is 0 or 1; b is 0 or 1; m is 0, 1, or 2; n is 0, 1, 2 or 3; r is 0 or1; s is 0 or 1; t is 0 or 1; R¹ and R² are independently selected from:H, (C₁-C₆)alkyl, aryl, heterocyclyl and (C₃-C₆)cycloalkyl, optionallysubstituted with one, two or three substituents selected from R⁷; R³ isselected from: 1) hydrogen; 2) C₁-C₁₀ alkyl; 3) C₁-C₁₀ alkyl-O—OR^(d),4) C₂-C₁₀ alkenyl-O—R^(d), 5) C₂-C₁₀ alkynyl-O—R^(d), 6)(C₁-C₆-alkylene)_(n)C₃-C₈ cycloalkyl-O—R^(d), 7) C₁-C₁₀akyl-(C═O)_(b)—NR^(c)R^(c)′, 8) C₂-C₁₀ alkenyl-(C═O)_(b)NR^(c)R^(c)′, 9)C₂-C₁₀ alkynyl-(C═O)_(b)NR^(c)R^(c)′, 10) (C16-alkylene)_(n)C₃-C₈cycloalkyl-C═O)_(b)NR^(c)R^(c)′, 11) C₁-C₁₀ alkyl-S(O)_(m)—R^(d), 12)C₂-C₁₀ alkenyl-S(O)_(m)—R^(d), 13) C₂-C₁₀ alkynyl-S(O)_(m)—R^(d), 14)(C₁-C₆-alkylene)_(n)C₃-C₈ cycloalkyl-S(O)_(m)—R^(d), said alkyl,alkenyl, alkynyl and cycloalkyl are optionally substituted with one ormore substituents selected from R⁶; R⁴ is independently selectedfrom: 1) (C═O)_(a)O_(b)C₁-C₁₀ alkyl, 2) (C═O)_(a)O_(b)aryl, 3) CO₂H, 4)halo, 5) CN, 6) OH, 7) O_(b)C₁-C₆ perfluoroalkyl, 8)O_(a)(C═O)_(b)NR⁸R⁹, 9) S(O)_(m)R^(a), 10) S(O)₂NR⁸R⁹, said alkyl, aryl,alkenyl, alkynyl, heterocyclyl, and cycloalkyl optionally substitutedwith one, two or three substituents selected from R⁷; R⁵ is selectedfrom: 1) hydrogen; 2) (C═O)_(a)O_(b)C₁-C₁₀ alkyl, 3) (C═O)_(a)O_(b)aryl,4) CO₂H, 5) halo, 6) CN, 7) OH, 8) O_(b)C₁-C₆ perfluoroalkyl, 9)O_(a)(C═O)_(b)NR⁸R⁹, 10) S(O)_(m)R^(a), 11) S(O)₂NR⁸R⁹, said alkyl,aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl optionallysubstituted with one, two or three substituents selected from R⁷; R⁶ isindependently selected from: 1) (C═O)_(a)O_(b)C₁-C₁₀ alkyl, 2)(C═O)_(a)O_(b)aryl, 3) C₂-C₁₀ alkenyl, 4) C₂-C₁₀ alkynyl, 5)(C═O)_(a)O_(b) heterocyclyl, 6) CO₂H, 7) halo, 8) CN, 9) OH, 10)O_(b)C₁-C₆ perfluoroalkyl, 11) O_(a)(C═O)_(b)NR⁸R⁹, 12) S(O)_(m)R^(a),13) S(O)₂NR⁸R⁹, 14) oxo, 15) CHO, 16) (N═O)R⁸R⁹, or 17)(C═O)_(a)O_(b)C₃-C₈ cycloalkyl, said alkyl, aryl, alkenyl, alkynyl,heterocyclyl, and cycloalkyl optionally substituted with one, two orthree substituents selected from R⁷; R⁷ is selected from: 1)(C═O)_(r)OS(C₁-C_(10)alkyl,) 2) O_(r)(C₁-C₃)perfluoroalkyl, 3) oxo, 4)OH, 5) halo, 6) CN, 7) (C₂C₁₀)alkenyl, 8) (C₂-10)alkynyl, 9)(C═O)_(r)O_(s)(C₃C₆)cycloalkyl, 10) (C═O)_(r)O_(s)(C₀-C₆)alkyene-aryl,11) (C═O)_(r)O_(s)(C₀-C₆)alkylene-heterocyclyl, 12)(C═O)_(r)O_(s)(C₀-C₆)alkylene-N(R^(b))₂, 13) C(O)R^(a), 14)(C₀-C₆)alkylene-CO₂R^(a), 15) C(O)H, 16) (C₀-C₆)alkylene-CO₂H, and 17)C(O)N(R^(b))₂, 18) S(O)_(m)R^(a), and 19) S(O)₂N(R^(b))₂; said alkyl,alkenyl, alkynyl, cycloalkyl, aryl, alkylene and heterocyclyl isoptionally substituted with up to three substituents selected fromR^(b), OH, (C₁-C₆)alkoxy, halogen, CO₂H, CN, O(C═O)C₁-C₆ alkyl, oxo, NO₂and N(R^(b))₂; R⁸ and R⁹ are independently selected from: 1) H, 2)(C═O)O_(b)C₁-C₁₀ alkyl, 3) (C═O)O_(b)C₃-C₈ cycloalkyl, 4)(C═O)O_(b)aryl, 5) (C═O)O_(b)heterocyclyl, 6) C₁-C₁₀ alkyl, 7) aryl, 8)C₂-C₁₀ alkenyl, 9) C₂-C₁₀ alkynyl, 10) heterocyclyl, 11) C₃-C₈cycloalkyl, 12) SO₂R^(a), and 13) (C═O)NR^(b) ₂, said alkyl, cycloalkyl,aryl, heterocylyl, alkenyl, and alkynyl is optionally substituted withone, two or three substituents selected from R⁷, or R⁸ and R⁹ can betaken together with the nitrogen to which they are attached to form amonocyclic or bicyclic heterocycle with 3-7 members in each ring andoptionally containing, in addition to the nitrogen, one or twoadditional heteroatoms selected from N, O and S, said monocyclic orbicyclic heterocycle optionally substituted with one, two or threesubstituents selected from R⁷; R¹⁰ is selected from: F and —CH₂F; R¹² isselected from: H and —C₂P, provided that when t is 1, R¹² is H; R^(ox)is absent or is oxo; R^(a) is independently selected from (C₁-C₆)alkyl,(C₃-C₆)cycloalkyl, aryl, or heterocyclyl, optionally substituted withone, two or three substituents selected from R⁷; R^(b) is independentlyselected from: H, (C₁-C₆)alkyl, aryl, heterocyclyl, (C₃-C₆)cycloalkyl,(C═O)OC₁-C₆ alkyl, (C═O)C₁-C₆ alkyl, (C═O)aryl, (C═O)heterocyclyl,(C═O)NR^(e)R^(e)′) or S(O)₂R^(a), optionally substituted with one, twoor three substituents selected from R⁷; R^(c) and R^(c)′ areindependently selected from: H, (C₁-C₆)alkyl, aryl, NH₂OH, OR^(a),—(C₁-C₆)alkyl-OH, —(C₁-C₆)alkyl-O—(C₁-C₆)alkyl, (C═O)OC₁-C₆ alkyl,(C═O)C₁-C₆ alkyl, (C═O)aryl, (C═O)heterocyclyl, (C═O)NR^(e)R^(e)′,S(O)₂R^(a) and —(C₁-C₆)alkyl-N(R^(b))₂, wherein the alkyl is optionallysubstituted with one, two or three substituents selected from R⁷; orR^(c) and R^(c)′ can be taken together with the nitrogen to which theyare attached to form a monocyclic or bicyclic heterocycle with 3-7members in each ring and optionally containing, in addition to thenitrogen, one or two additional heteroatoms selected from N, O and S,said monocyclic or bicyclic heterocycle optionally substituted with one,two or three substituents selected from R⁷; R^(d) is selected from: H,(C₁-C₆)alkyl, —C₂-C₆)alkyl-OH, —C₁-C₆)alyl-O—(C₁-C₆)alkyl and—C₁-C₆)alkyl-N(R^(b))₂, wherein the alkyl is optionally substituted withone, two or three substituents selected from R⁷; R^(e) and R^(e)′ areindependently selected from: H (C₁-C₆)alkyl, aryl, heterocyclyl and(C₃-C₆)cycloalkyl, optionally substituted with one, two or threesubstituents selected from R⁷; or R^(e) and R^(e)′ can be taken togetherwith the nitrogen to which they are attached to form a monocyclic orbicyclic heterocycle with 3-7 members in each ring and optionallycontainig, in addition to the nitrogen, one or two additionalheteroatoms selected from N, O and S, said monocyclic or bicyclicheterocycle optionally substituted with one, two or three substituentsselected from R⁷.
 3. The compound according to claim 2 of Formula III:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:a is 0 or 1; b is 0 or 1; m is 0, 1, or 2; n is 0, 1 or 2; r is 0 or 1;s is 0 or 1; R¹ and R² are independently selected from: H, (C₁-C₆)alkyl,aryl and (C₃-C₆)cycloalcyl, optionally substituted with one, two orthree substituents selected from R⁷; R⁴ is independently selectedfrom: 1) halo, 2) OH, 3) O_(b)C₁-C₆ perfluoroalkyl, R⁵ is selectedfrom: 1) hydrogen, 2) halo, 3) OH, 4) O_(b)C₁-C₆ perfluoroalIyl, R⁷ isselected from: 1) (C═O)_(r)OS(C₁-C₁₀)alkyl, 2)O_(r)(C₁-C₃)perfluoroaikyl, 3) oxo, 4) OH, 5) halo, 6) CN, 7)(C₂-C₁₀)alkenyl, 8) (C₂-C₁₀)alkynyl, 9) (C═O)_(r)O_(s)(C₃-C₆)cycloalkyl,10) (CO)_(r)O_(s)(C₀-C₆)alkylene-aryl, 11)(C═O)_(r)O_(s)(C₀-C₆)alkylene-heterocyclyl, 12)(C═O)_(r)O_(s)(C₆)alene-N(R^(b))₂, 13) C(O)R^(a), 14)(C₀-C₆)alkylene-CO₂R^(a), 15) C(O)H, 16) (C₀-C₆)alkylene-CO₂H, and 17)C(O)N(R^(b))₂, 18) S(O)_(m)R^(a), and 19) S(O)₂N(R^(b))₂; said alkyl,alkenyl, alkynyl, cycloalkyl, aryl, alkylene and heterocyclyl isoptionally substituted with up to three substituents selected fromR^(b), OH, (C₁-C₆)alkoxy, halogen, CO₂H, CN, O(C═O)C₁-C₆ alkyl, oxo, NO₂and N(R^(b))₂; R⁸ and R⁹ are independently selected from: 1) H, 2)(C═O)O_(b)C₁-C₁₀alkyl, 3) (C═O)O_(b)C₃-C₈ cycloalkyl, 4) (C═O)O_(b)aryl,5) (C═O)O_(b)heterocyclyl, 6) C₁-C₁₀ alkyl, 7) aryl, 8) C₂-C₁₀ alkenyl,9) C₂-C₁₀ alkynyl, 10) heterocyclyl, 11) C₃-C₈ cycloalkyl, 12) SO₂R^(a),and 13) (C═O)NR^(b) ₂, said alkyl, cycloalkyl, aryl, heterocylyl,alkenyl, and alkynyl is optionally substituted with one, two or threesubstituents selected from R⁷, or R⁸ and R⁹ can be taken together withthe nitrogen to which they are attached to form a monocyclic or bicyclicheterocycle with 3-7 members in each ring and optionally containing, inaddition to the nitrogen, one or two additional heteroatoms selectedfrom N, O and S, said monocyclic or bicyclic heterocycle optionallysubstituted with one, two or three substituents selected from R⁷; R^(a)is independently selected from: (C₁-C₆)alkyl, (C₃-C₆)cycloalkyl, aryl,or heterocyclyl, optionally substituted with one, two or threesubstituents selected from R⁷; R^(b) is independently selected from H,(C₁-C₆)alkyl, aryl, heterocyclyl, (C₃-C₆)cycloalkyl, (C═O)OC₁-C₆ alkyl,(C═O)C₁-C₆ alkyl, (C═O)aryl, (C═O)heterocyclyl, (C═O)NR^(e)R^(e)′ orS(O)₂R^(a), optionally substituted with one, two or three substituentsselected from R⁷; R^(c) and R^(c)′are independently selected from: H,(C₁-C₆)alkyl, aryl, NH₂, OH, OR^(a), —(C₁-C₆)alkyl-OH,—(C₁-C₆)alkyl-O—(C₁-C₆)alkyl, (C═O)OC₁-C₆ alkyl, (C═O)C₁-C₆ alkyl,(C═O)aryl, (C═O)heterocyclyl, (C═O)NR^(e)R^(e)′, S(O)₂R^(a) and—C₁-C₆)alkyl-N(R^(b))₂, wherein the alkyl is optionally substituted withone, two or three substituents selected from R⁷; or R^(c) and R^(c)′ canbe taken together with the nitrogen to which they are attached to form amonocyclic or bicyclic heterocycle with 3-7 members in each ring andoptionally containing, in addition to the nitrogen, one or twoadditional heteroatoms selected from N, O and S, said monocyclic orbicyclic heterocycle optionally substituted with one, two or threesubstituents selected from R⁷; R^(e) and R^(e)′are independentlyselected from: H, (C₁-C₆)alkyl, aryl, heterocyclyl and(C₃-C₆)cycloalkyl, optionally substituted with one, two or threesubstituents selected from R⁷; or R^(e) and R^(e)′ can be taken togetherwith the nitrogen to which they are attached to form a monocyclic orbicyclic heterocycle with 3-7 members in each ring and optionallycontaining, in addition to the nitrogen, one or two additionalheteroatoms selected from N, O and S, said monocyclic or bicyclicheterocycle optionally substituted with one, two or three substituentsselected from R⁷.
 4. The compound according to claim 3 of the formulaIV:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:a is 0 or 1; b is 0 or 1; m is 0, 1, or 2; r is 0 or 1; s is 0 or 1; R¹and R² are independently selected from: H and (C₁-C₆)alkyl, optionallysubstituted with one, two or three substituents selected from R⁷; R⁴ isindependently selected from 1) halo, 2) OH, 3) O_(b)C₁-C₆perfluoroalkyl, R⁷ is selected from: 1) (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, 2)O_(r)(C₁-C₃)perfluoroalkyl, 3) oxo, 4) OX, 5) halo, 6) CN, 7)(C₂-C₁₀)alkenyl, 8) (C₂-C₁₀)alkynyl, 9) (C═O)_(r)O_(s)(C₃-C₆)cycloalkyl,10) (C═O)_(r)O_(s)(C₀-C₆)alkylene-aryl, 11)(C═O)_(r)O_(s)(C₀-C₆)alkylene-heterocyclyl, 12)(C═O)_(r)O_(s)(C₀-C₆)aklene-N(R^(b))₂, 13) C(O)R^(a), 14)(C₀-C₆)alkylene-CO₂R^(a), 15) C(O)H, 16) (C₀-C₆)alkylene-CO₂H, and 17)C(O)N(R^(b))₂, 18) S(O)_(m)R^(a), and 19) S(O)₂N(R^(b))₂; said alkyl,alkenyl, alkynyl, cycloalkyl, aryl, alkylene and heterocyclyl isoptionally substituted with up to three substituents selected fromR^(b), OH, (C₁-C₆)alkoxy, halogen, CO₂H, CN, O(C═O)C₁-C₆ alkyl, oxo, NO₂and N(R^(b))₂; R⁸ and R⁹ are independently selected from: 1) H, 2)(C═O)O_(b)C₁-C₁₀ alkyl, 3) (C═O)O_(b)C₃-C₈ cycloalkyl, 4)(C═O)O_(b)aryl, 5) (C═O)O_(b)heterocyclyl, 6) C₁-C₁₀ alkyl, 7) aryl, 8)C₂-C₁₀ alkenyl, 9) C₂-C₁₀ alkynyl, 10) heterocyclyl, 11) C₃-C₈cycloalkyl, 12) SO₂R^(a), and 13) (C═O)NR^(b) ₂, said alkyl, cycloalkyl,aryl, heterocylyl, alkenyl, and alkynyl is optionally substituted withone, two or three substituents selected from R⁷, or R⁸ and R⁹ can betaken together with the nitrogen to which they are attached to form amonocyclic or bicyclic heterocycle with 3-7 members in each ring andoptionally containing, in addition to the nitrogen, one or twoadditional heteroatoms selected from N, O and S, said monocyclic orbicyclic heterocycle optionally substituted with one, two or threesubstituents selected from R⁷; R^(a) is independently selected from:(C₁-C₆)alkyl, (C₃-C₆)cycloalkyl, aryl, or heterocyclyl, optionallysubstituted with one, two or three substituents selected from R⁷; R^(b)is independently selected from: H, (C₁-C₆)alkyl, aryl, heterocyclyl,(C₃-C₆)cycloalkyl, (C═O)OC₁-C_(C) ₆ alkyl, (C═O)C₁-C₆ alkyl, (C═O)aryl,(C═O)heterocyclyl, (C═O)NR^(e)R^(e)′ or S(O)₂R^(a), optionallysubstituted with one, two or three substituents selected from R⁷; R^(c)and R^(c)′ are independently selected from H, (C₁-C₆)alkyl, aryl, NH₂OH,OR^(a), —(C₁-C₆)alkyl-OH, —(C₁-C₆)alkyl-O—(C₁-C₆)alkyl, (C═O)OC₁-C₆alkyl, (C═O)C₁-C₆ alkyl, (C═O)aryl, (C═O)heterocyclyl,(C═O)NR^(e)R^(e)′, S(O)₂R^(a) and —C₁-C₆)alkyl-N(R^(b))₂, wherein thealkyl is optionally substituted with one, two or three substituentsselected from R⁷; or R^(c) and R^(c)′ can be taken together with thenitrogen to which they are attached to form a monocyclic or bicyclicheterocycle with 3-7 members in each ring and optionally containing, inaddition to the nitrogen, one or two additional heteroatoms selectedfrom N, O and S, said monocyclic or bicyclic heterocycle optionallysubstituted with one, two or three substituents selected from R⁷; R^(e)and R^(e)′ are independently selected from: H, (C₁-C₆)alkyl, aryl,heterocyclyl and (C₃-C₆)cycloallyl, optionally substituted with one, twoor three substituents selected from R⁷; or R^(e) and R^(e)′ can be takentogether with the nitrogen to which they are attached to form amonocyclic or bicyclic heterocycle with 3-7 members in each ring andoptionally containing, in addition to the nitrogen, one or twoadditional heteroatoms selected from N, O and S, said monocyclic orbicyclic heterocycle optionally substituted with one, two or threesubstituents selected from R⁷.
 5. A compound selected from:(2S)-4-(2,5-Difluorophenyl)-N-[(3R,4R)-4-fluoropyrrolidin-3-yl]-2-(hydroxymethyl)N-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide(2S)-4-(2,5-Difluorophenyl)-N-[(3S,4S)-4-fluoropyrrolidin-3-yl]-2-hydroxymethyl)N-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide(2S)-4-(2,5-Difluorophenyl)-N-[(3R,4R)-4-fluoro-1-methylpyrrolidin-3-yl]-2-(hydroxymethyl)N-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide(2S)4-2,5-Difluorophenyl)-N-[(3S,4S)-4-fluoro-1-methylpyrrolidin-3-yl]-2-(hydroxymethyl)N-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide(2S)-4-2,5-Difluorophenyl)-N-[(3S,5S)-5-(fluoromethyl)-pyrrolidin-3-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5dihydro-1H-pyrrole-1-carboxaride(2S)-4-2,5-Difluorophenyl)-N-[(3S,5S)-5-fluoromethyl)-1-methylpyrrolidin-3-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide(2S)-4-2,5-Difluorophenyl)-N-[(3S,5R)-5-(fluoromethyl)-pyrrolidin-3-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide(2S)-4-2,5-Difluorophenyl)-N-[(3S,5R)-5-(fluoromethyl)-1-methylpyrrolidin-3-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamideor a pharmaceutically acceptable salt or stereoisomer thereof.
 6. Apharmaceutical composition that is comprised of a compound in accordancewith claim 1 and a pharmaceutically acceptable carrier.
 7. A method ofusing the compound according to claim 1 for the preparation of amedicament useful in treating or preventing cancer in a mammal in needof such treatment.
 8. A method of using the compound according to claim1 for the preparation of a medicament useful in treating or preventingcancer in a mammal in need of such treatment, wherein the cancer isselected from histiocytic lymphoma, lung adenocarcinoma, small cell lungcancers, pancreatic cancer, gioblastomas and breast carcinoma.
 9. Amethod of using the compound according to claim 1 for the preparation ofa medicament useful for modulating mitotic spindle formation in a mammalin need of such treatment.